Lymphocyte surface receptor that binds caml and methods of use thereof

ABSTRACT

A novel lymphocyte receptor protein, its DNA sequence, and its role in the calcium activation pathway is described. The protein, or genetically engineered constructs encoding it, is shown to increase lymphocyte response, and to identify ligands of the protein receptor. Antibodies to the proteins of the invention are generated for diagnostic therapeutics. The protein and DNA can also be used for diagnostic purposes and for identifying agents for modulating the calcium induced activation pathway. A particular advantage of the present invention is that it provides lymphocyte activation of receptor found on all B cells, but only on a subset of T cells. The receptor can thus be targeted to specifically regulate B cell responses without affecting mature T cell activity. Such targeting specificity is always advantageous, particularly where an increase or decrease of antibody production is desired, e.g., during an infection (increase) or to avoid immune complex deposition complications (rheumatoid arthritis, glomerulonephritis, and other auto immune conditions).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/079,418, filed Mar. 14, 2005, which is a divisional of U.S.application Ser. No. 10/293,816, filed Nov. 12, 2002; which is adivisional of U.S. application Ser. No. 09/782,857, filed Feb. 14, 2001,now U.S. Pat. No. 6,500,428, issued Dec. 31, 2002; which is a divisionalof U.S. application Ser. No. 09/290,333, filed Apr. 12, 1999, now U.S.Pat. No. 6,316,222, issued Nov. 13, 2001; which is a divisional of U.S.application Ser. No. 08/810,572, filed Mar. 3, 1997, now U.S. Pat. No.5,969,102, issued Oct. 19, 1999, each of which are hereby incorporatedin their entirety by reference herein.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The research leading to the present invention was supported in part bythe Cancer Center CORE Grant CA-21765 from the National Institutes ofHealth. The government may have certain rights in the present invention.Support for this invention was also provided by the AMERICAN LEBANESESYRIAN ASSOCIATED CHARITIES, the American Cancer Society Grant Bt-234,and the James McDonnell Foundation Grant JSMF 93-40-03.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named340547SEQLIST.txt, created on Feb. 7, 2008, and having a size of 10.1 KBand is filed concurrently with the specification. The sequence listingcontained in this ASCII formatted document is part of the specificationand is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to the regulation of transcription inlymphocytes, proteins involved therein, antibodies thereof, nucleicacids that encode the proteins and uses of the nucleic acids, antibodiesand proteins.

BACKGROUND OF THE INVENTION

Investigators are only beginning to unravel the mechanisms that controlthe cellular response to extrinsic factors. One basic feature of many ofsuch mechanisms is the initial binding of an extrinsic factor, e.g., aligand, to a cell surface membrane protein, i.e., a receptor. Thebinding of a ligand to its receptor usually effects a cellular changethrough a cascade of events. These events commonly involve otherproteins, such as protein kinases, protein phosphatases, JAK proteins,Stat proteins, and/or G-proteins. In addition, there is generally arequirement for a transcription factor to bind to a specific DNAregulatory sequence in the nucleus of the cell, and thereby initiate thetranscription of one or more particular genes.

Other factors are often involved. In antigen-stimulated lymphocyteactivation, for example, calcium (Ca²⁺) influx is also necessary for theultimate initiation of DNA transcription. The increased cytoplasmiccalcium concentration may originate as an external influx or a releaseof internal stores. Increased calcium concentration which activates thecalcium-dependent protein phosphatase calcineurin acts in conjunctionwith other agents to signal the initiation of transcription. It is clearthat the pathway involving calcium influx is essential to a number ofprocesses involved with activation and proliferation of cells.

Intracellular calcium levels play a major function in a number ofdifferent cell types involving a number of different activities. Inaddition to the induction of gene transcription by calcium influx, manyother calcium-dependent events, such as those which occur during musclecontraction (both cardiac and skeletal), vesicle degranulation (such asin the response of neutrophils and macrophages to infection, or basophilresponse to antigen stimulation, or release of acetylcholine byneurons), and closure of intracellular gap junctions offer opportunitiesfor cellular regulation. The cell cycle can also involve fluxes ofcalcium. Intracellular chelators which block changes in intracellularcalcium concentration can block the cell cycle from progressing, therebyarresting cell division. [Rabinovich et al. (1986) J. of Immunol.137:952-961]. Therefore, regulation of calcium can be effective inmodulating cell division in normal and diseased cells.

Lymphocytes are a primary component of the cellular arm of the immunesystem. Activation of one particular type of lymphocyte, a T-cell, canresult through the stimulation of a T-cell receptor by e.g., the bindingof a T-cell receptor (TCR) to an antigen presented by anantigen-presenting cell. This stimulation results in the activation aC²⁺-dependent phosphatase, calcineurin. Activated calcineurin, in turn,activates NF-AT, a lymphocyte specific transcription factor thattogether with a companion transcription factor, AP-1, effects theexpression of the inducible T-cell growth factor, interleukin-2 (IL-2).Activation of AP-1 is a calcium-independent process that involvesprotein kinase C, and can be experimentally achieved with the additionof phorbol myristate acetate (PMA). The immunosuppressant drugcyclosporin A (CsA) binds to and inhibits the prolyl isomerase activityof cyclophilin and the resulting drug-isomerase complex inactivatescalcineurin, by a direct interaction near the active site of the enzyme.[Liu et al. (1991) Cell 66:807-15]; [Clipstone and Crabtree (1992)Nature 357:695-7]; [O'Keefe et al. (1992) Nature 357:692-4]. NF-κB is athird key transcription factor which is important in the activation oflymphocytes and which is activated following the stimulation of theT-cell or B-cell antigen receptor.

Another protein associated with the calcium signaling pathway inlymphocytes is the recently identified calcium-signal modulatingcyclophilin ligand (CAML) [Bram, R. J. and Crabtree, G. R., DNA EncodingCalcium-Signal Modulating Cyclophilin Ligand, U.S. Pat. No. 5,523,227,issued Jun. 4, 1996, hereby incorporated by reference in its entirety].CAML binds cyclophilin B with reasonable specificity, i.e., CAML doesnot bind cyclophilin A or C. Unlike the cyclosporin A-cyclophilincomplex, however, the CAML-cyclophilin B complex does not directly bindto calcineurin. Thus CAML appears to affect calcineurin through itsregulation of Ca²⁺ influx. As expected, CsA can indirectly block theactivating effect of CAML on transcription, by inhibiting calcineurin.In addition, CAML appears to have no effect on the activation of AP-1,and so the CAML-dependent activation of NF-AT experimentally requiresthe addition of PMA.

CAML acts downstream from an extrinsic signal but upstream fromcalcineurin. The location of CAML in cytoplasmic vesicles suggests thatit can regulate Ca²⁺ influx by modulating intracellular Ca²⁺ release.However, there remains a need to determine the natural factor (orfactors) that communicate the external signal to the cellular CAML.Further, there is a need to understand how CAML interacts with thisfactor in order to learn how to better control the important cellularprocesses that CAML helps to regulate. A different class of signalingmolecule is the TNFR family of cell surface receptors [Smith et al.(1994) Cell 76:959-62]. These receptors initiate intracellular signalsleading to the onset of cell growth, death, or gain of effectorfunction.

SUMMARY OF THE INVENTION

A novel lymphocyte receptor, its DNA sequence, and its role in thecalcium activation pathway is described. The protein, or geneticallyengineered constructs encoding it, can be used to enhance lymphocyteresponse, or to identify ligands of the protein receptor. The soluble,extracellular domain can be used to inhibit cellular activation.Antibodies to the protein can be generated for diagnostic or therapeuticuses. The protein and DNA may also be used for diagnostic purposes andfor identifying agents for modulating the calcium induced activationpathway. Knowledge of the coding sequence allows for manipulation ofcells to elucidate the mechanism of which CAML is a part.

A particular advantage of the present invention is that it provideslymphocyte activation of a receptor found on all B cells, but only on asubset of T cells. The receptor can thus be targeted to specificallyregulate B cell responses without affecting mature T cell activity. Suchtargeting specificity is always advantageous, particularly where anincrease or decrease of antibody production independent of cellularimmune responses is desired, e.g., during an infection (increase) or toavoid immune complex deposition complications (rheumatoid arthritis,glomerulonephritis, and other autoimmune conditions).

Crosslinking the novel cell surface receptor of the present inventionactivates B cells and some populations of T cells. Activation of thesecells increases the immune system activity. On the other hand, blockingor inhibiting the novel cell surface receptor of the present inventioncan result in immunosuppression. Depending on the endogenous level ofactivation of the receptor, which can be evaluated using the antibodiesor nucleic acids of the invention, receptor activity can be enhanced orsuppressed to achieve a desired outcome. Either activating or inhibitingthe function of the novel cell surface receptor of the present inventioncan be used to treat cancers of T and B cells.

The present invention includes an isolated Transmembrane Activator andCAML-Interactor (TACI) protein that functions as a cell surfacesignaling protein and comprises an extracellular domain, a membranespanning segment, and a cytoplasmic domain. In one embodiment, the TACIprotein is a plasma membrane receptor in which the extracellular domainresides at the N-terminal portion of the protein and the cytoplasmicdomain resides at the C-terminal portion of the protein. The N-terminalportion of the TACI protein functions as a binding site for a ligandthat stimulates the activation of the cell by inducing the binding ofthe C-terminal portion of the TACI protein to the N-terminal domain ofCAML. Since CAML is an integral membrane protein that is localized tocytoplasmic vesicles, the TACI protein is a plasma membrane receptorthat directly interacts with an intracellular organelle in lymphocytes.

In one embodiment, the monomeric form of the isolated TACI proteinconsists of about 295 amino acids. In a preferred embodiment themonomeric form of a Transmembrane Activator and CAML Interactor (TACI)protein contains 280 to 310 amino acids. In more preferred embodimentsthe monomeric form of a TACI protein contains 290 to 296 amino acids. Ina specific embodiment exemplified infra, the monomeric form of a TACIprotein contains 293 amino acids.

One embodiment of the isolated TACI protein contains two TNFRsuperfamily cysteine-rich repeats [Bairoch (1993) Nucl. Acids Res.21:3097-3103]. In a preferred embodiment, a TACI protein that isappropriately stimulated, in situ, such as by a ligand or an anti-TACIantibody, initiates the activation of a transcription factor through thecombination of a Ca²⁺-dependent pathway and a Ca²⁺-independent pathway.

The present invention includes an isolated nucleic acid that consists ofat least 18 nucleotides of a nucleotide sequence that has at least 60%similarity with SEQ ID NO:1, or alternatively at least 60% similaritywith the coding sequence of SEQ ID NO:1. The nucleotide sequence encodesa TACI protein which has a binding affinity for CAML. In one suchembodiment the isolated nucleic acid encodes a TACI protein.

In a preferred embodiment of the present invention the nucleotidesequence has at least 75% similarity with SEQ ID NO:1, or has at least75% similarity with the coding sequence of SEQ ID NO:1. In a morepreferred embodiment, the nucleotide sequence has at least 90%similarity with SEQ ID NO:1, or has at least 90% similarity with thecoding sequence of SEQ ID NO:1. In an even more preferred embodiment,the nucleotide sequence has between 95-98% similarity with SEQ ID NO:1,or has between 95-98% similarity with the coding sequence of SEQ IDNO:1. In a particular embodiment the nucleotide sequence is SEQ ID NO:1.In a related embodiment, the nucleotide sequence consists of the codingsequence of SEQ ID NO:1. In a specific embodiment, exemplified infra,the isolated nucleic acid has the nucleotide sequence of SEQ ID NO:1. Ina related embodiment, the isolated nucleic acid consists of the codingsequence of SEQ ID NO:1.

In another related embodiment the present invention includes an isolatednucleic acid which contains at least 18 nucleotides and hybridizes toSEQ ID NO:1, or more particularly hybridizes to the coding sequence ofSEQ ID NO:1. In one such embodiment, the hybridization is performedunder moderate stringency. In another embodiment, the hybridization isperformed under standard hybridization conditions. In yet a thirdembodiment, the hybridization is performed under stringent hybridizationconditions.

In still another related embodiment the present invention includes anisolated nucleic acid which contains at least 18 nucleotides of anucleotide sequence that encodes a TACI protein having an amino acidsequence of either SEQ ID NO:2, or SEQ ID NO:2 with one or moreconservative substitutions. In one such embodiments of this type, theisolated nucleic acid encodes an N-terminal fragment of the TACI proteincorresponding to the extracellular domain. In another embodiment, theisolated nucleic acid encodes a C-terminal fragment of the TACI proteinthat is sufficient to bind to the N-terminal 146 amino acids of CAML. Inyet another embodiment, the isolated nucleic acid encodes thetransmembrane portion of the TACI protein. In still another embodiment,the isolated nucleic acid encodes the full-length TACI protein.

In a preferred embodiment of the present invention, the isolated nucleicacid consists of at least 24 nucleotides. In a more preferredembodiment, the isolated nucleic acid consists of at least 30nucleotides. In an even more preferred embodiment, the isolated nucleicacid consists of at least 36 nucleotides. Oligonucleotides of theinvention can be used as nucleic acid probes, PCR primers, antisensenucleic acids, and the like, for diagnostic and therapeutic purposes.

In one embodiment of the present invention, an isolated nucleic acid(SEQ ID NO:3) encodes a C-terminal fragment of the TACI protein that issufficient to bind to the N-terminal 146 amino acids of CAML. In oneparticular embodiment of this type, the C-terminal fragment containsabout 126 amino acids. In another embodiment of this type the C-terminalfragment has an amino acid sequence of either SEQ ID NO:4, or SEQ IDNO:4 with one or more conservative substitutions.

In another embodiment, an isolated nucleic acid of the invention encodesan N-terminal fragment (SEQ ID NO:5) of the CAML-binding proteincorresponding to the extracellular domain. In a particular embodiment ofthis type the N-terminal fragment has an amino acid sequence of eitherSEQ ID NO:6, or SEQ ID NO:6 with one or more conservative substitutions.

In a preferred embodiment, the isolated nucleic acid encodes a TACIprotein that has a binding affinity for CAML. When such a TACI proteinis appropriately stimulated, in situ, it initiates activation of atranscription factor through the combination of a Ca²⁺-dependent pathwayand a Ca²⁺-independent pathway. In a more preferred embodiment, theisolated nucleic acid encodes a TACI protein having the amino acidsequence of SEQ ID NO:2.

The present invention also includes a DNA construct comprising anisolated nucleic acid of the present invention that is a recombinant DNAoperatively linked to an expression control sequence. The expressioncontrol sequence can be selected from the group consisting of the earlyor late promoters of SV40 or adenovirus, the lac system, the trp system,the TAC system, the TRC system, the major operator and promoter regionsof phage λ, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase, the promoters of acid phosphatase and thepromoters of the yeast α-mating factors.

In a preferred embodiment, the expression control sequence is either astandard tet inducible promoter, a metallothionein promoter, or anecdysone promoter. In a more preferred embodiment, the expressioncontrol sequence is the SR_(α), promoter.

The present invention also includes a unicellular host transformed witha recombinant DNA construct of the present invention. In one embodimentthe unicellular host is a prokaryote. In another embodiment theunicellular host is a eukaryote. Preferably the eukaryotic host is amammalian cell, for example, a COS, CHO or Jurkat T cell, which could beuseful for evaluating activity of the TACI protein or to identifymodulatory agents.

The present invention includes the isolated polypeptides encoded by thenucleic acids of the present invention, fragments thereof, and fusionproteins thereof. In one embodiment, the polypeptide fragment consistsof an N-terminal fragment of the TACI protein corresponding to theregulatory extracellular domain. In a particular embodiment theN-terminal fragment has an amino acid sequence of SEQ ID NO:6 or SEQ IDNO:6 with one or more conservative substitutions.

In another embodiment, the polypeptide fragment consists of a C-terminalfragment of the TACI protein that is sufficient to bind to theN-terminal 146 amino acids of CAML. In one such embodiment, theC-terminal fragment contains 95 to 130 amino acids. In a specificembodiment, the C-terminal fragment contains the C-terminal 126 aminoacids of SEQ ID NO:2. In an alternative embodiment the C-terminalfragment of the TACI protein contains about 110 amino acids. In apreferred embodiment of this type, the C-terminal fragment contains 107amino acids and has an amino acid sequence of SEQ ID NO:4.

The present invention also includes the preparation of a recombinantform of the extracellular portion of a TACI protein, thereby creating adominant-negative or blocking reagent. This component intercepts thenormal endogenous ligands which serve to crosslink and activate the TACIprotein. Administration of such a polypeptide acts to suppress theimmune system. Such administration is useful in the treatment orprevention of autoimmune disease or graft-rejection or graft-vs-hostdisease following transplantation.

A chimeric TACI protein of the invention may be a protein that isgenerated by joining the extracellular domain of another receptormolecule with a transmembrane domain and the intracellular domain of aTACI protein. In another embodiment, the extracellular domain of a TACIprotein can be joined with a transmembrane domain and an intracellulardomain of another receptor molecule. The transmembrane domain can be thetransmembrane domain of a TACI protein, the transmembrane domain of theother receptor, or a different transmembrane domain. Preferably, thetransmembrane domain is from the same protein component of the chimeraas the extracellular domain.

In a preferred embodiment the polypeptide is a TACI protein encoded by anucleic acid of the present invention that has a binding affinity forCAML and when appropriately stimulated, in situ, initiates activation ofa transcription factor through the combination of a Ca²⁺-dependentpathway and a Ca²⁺-independent pathway.

The present invention also includes antisense nucleic acids thathybridize under physiological conditions to the mRNAs that encode theTACI proteins of the present invention. Such antisense nucleic acids maybe RNA transcribed from an antisense gene, or RNA or DNA producedexogenously (whether by expression or chemical synthesis). Preferably, asynthetic antisense nucleic acid is prepared with non-naturallyoccurring bonds to prevent its rapid hydrolysis and thus increase itseffective half-life.

A knockout animal is also part of the present invention. The knockoutanimal comprises a first and second allele which each naturally encodeand express functional TACI protein but in which at least one of the twoalleles is defective and thereby prevents the animal from expressing anadequate amount of the TACI protein. In one embodiment of this type, thefirst allele contains a defect that prevents the animal from expressingany functional TACI protein. In a preferred embodiment, a knockoutanimal contains both a defective first allele and a defective secondallele. These defective alleles prevent the animal from expressingfunctional TACI protein. In a preferred embodiment, the knockout animalis a knockout mouse.

The present invention also includes antibodies to all of the nucleicacids and polypeptides of the present invention. In a specificembodiment, the antibody is prepared against the TACI protein having anamino acid sequence of SEQ ID NO:2, or an antigenic fragment thereof.The antibodies of the present invention can be either monoclonalantibodies or polyclonal antibodies. In one embodiment, the antibody isa monoclonal antibody that is a chimeric antibody.

An immortal cell line that produces a monoclonal antibody of the presentinvention is also part of the present invention. In a specificembodiment of this immortal cell line, the monoclonal antibody isprepared against the TACI protein having an amino acid sequence of SEQID NO:2 or an antigenic fragment thereof.

The present invention also includes an N-terminal fragment of CAML thatis sufficient to bind to the C-terminal 126 amino acid fragment ofTACI-1. In one such embodiment, the N-terminal fragment of CAML contains146 amino acids. This N-terminal fragment of CAML can serve as aninhibitor of TACI-CAML binding.

The present invention includes methods of making TACI proteins,fragments thereof and fusion proteins thereof. In one embodiment themethod comprises introducing an expression vector comprising a nucleicacid encoding a polypeptide that is a TACI protein, or a fragmentthereof, or a fusion protein thereof, into a host cell and expressingthe encoded polypeptide. In a preferred embodiment the expressedpolypeptide has a binding affinity for CAML. In a more preferredembodiment the polypeptide, when appropriately stimulated, in situ,initiates activation of a transcription factor through the combinationof a Ca²⁺-dependent and a Ca²⁺-independent pathway. In the mostpreferred embodiment of this type the expressed polypeptide is a TACIprotein having an amino acid sequence of SEQ ID NO:2.

Methods of purifying the expressed polypeptides encoding TACI proteins,fragments thereof and fusion proteins thereof, are also part of thepresent invention, as are the purified expressed polypeptidesthemselves.

The present invention also includes methods for identifying a ligand fora TACI protein. One embodiment of such a method comprises contacting theN-terminal extracellular polypeptide of a TACI protein with a candidatemolecule and detecting the binding of the N-terminal extracellularpolypeptide with the candidate molecule. The binding of the N-terminalextracellular polypeptide with the candidate molecule indicates that thecandidate molecule is ligand.

In an alternative method for identifying a ligand for a TACI protein, afunctional TACI protein is used. In preferred embodiments of this typethe functional TACI protein is TACI-1. The binding of the functionalTACI protein with the candidate molecule indicates that the candidatemolecule is a ligand. In one such embodiment, binding of the candidatemolecule to the functional TACI protein is determined by detectingcellular activation as a function of the level of activation of the AP-1pathway. In another embodiment, binding of the candidate molecule to thefunctional TACI protein is determined by detecting cellular activationas a function of the level of activation of the CAML pathway.

In another embodiment, binding of the candidate molecule to thefunctional TACI protein is determined by detecting cellular activationas a function of the level of the concentration of the NF-ATtranscription factor. In still another embodiment, binding of thecandidate molecule to the functional TACI protein is determined bydetecting cellular activation as a function of the level of activationof the NF-κB pathway. In yet another embodiment, binding of thecandidate molecule to the functional TACI protein is determined bydetecting cellular activation as a function of the level of theactivation of NF-AT. In this case, the level of activation of NF-AT canbe determined by methods including demonstrating cytoplasm to nucleartranslocation of NF-AT; the relative dephosphorylation of NF-AT; and/orby NF-AT-dependent transcription.

In preferred embodiments, more than one of the above determinations ofcellular activation is made, and the candidate molecule is identified asa ligand when all the determinations made indicate the binding of thecandidate molecule to the TACI protein. In the most preferredembodiment, all of the above determinations of cellular activation aremade and the candidate molecule is identified as a ligand when all ofthese determinations indicate that the candidate molecule binds to theTACI protein.

Methods for identifying a ligand for a TACI protein may be performed ina large number of expression systems in the TACI protein can beexpressed. One embodiment employs the use of a yeast two-hybridexpression system using the TACI protein as “bait.” In anotherembodiment, interaction cloning from E. coli expression-libraries may beemployed. In yet another embodiment, functional expression cloning inmammalian cells of the TACI protein can be utilized. In a preferredembodiment, the mammalian cells are B-cell derived lines such asBurkitt's Lymphoma, EBV-immortalized cell lines, or multiple myelomacell lines. In a more preferred embodiment of this type, the TACIprotein is expressed in Jurkat T cells containing a reporter gene undercontrol of an NF-AT promoter. In one such embodiment, the reporter geneencodes secreted alkaline phosphatase (SEAP) as the marker.

The present invention also includes methods of screening for animmunosuppressant drug that inhibits the activation of B cells to agreater extent than it inhibits the activation of mature T cells. Inpreferred embodiments of this type, the immunosuppressant drug inhibitsthe activation of B cells, but does not inhibit the activation of matureT cells. Such methods may be performed in transformed T cells, such as aJurkat T cell, which can be genetically manipulated to express the TACIprotein; or in B cells that naturally express the TACI protein. Thepresent invention also includes the immunosuppressant drugs identifiedwhich inhibit the activation of B cells, but not the activation ofmature T cells.

The present invention includes methods of identifying animmunosuppressant drug that selectively blocks the action of Blymphocytes without effecting mature T lymphocytes. One such embodimentcomprises contacting a first lymphocyte with a potential drug, whereinthe first lymphocyte contains a TACI protein and a first marker protein.The first marker protein is transcribed when the TACI protein isstimulated in the absence of a candidate drug. The TACI protein isstimulated, and the first marker protein is detected under conditions inwhich if it is transcribed, it is detectable. A potential drug isselected as a candidate drug when the first marker protein cannot bedetected. Next, a second lymphocyte is contacted with the candidatedrug, wherein the second lymphocyte contains a T cell receptor, and asecond marker protein that is transcribed when the T cell receptor isstimulated either in the absence or the presence of theimmunosuppressant drug. The T cell receptor is stimulated and the secondmarker protein is detected under conditions in which if it istranscribed, it is detectable. A candidate drug is identified as animmunosuppressant drug when the second marker protein is detected, sincethe immunosuppressant drug interferes with the pathway (or aspectthereof) involving the TACI protein but not the pathway (or aspectthereof) involving the T cell receptor.

In one embodiment, the first and second lymphocytes are Jurkat T cellsthat have been modified to express a TACI protein. In one suchparticular embodiment the method comprises contacting a first Jurkat Tcell with a potential drug, wherein the first Jurkat T cell has beengenetically engineered to express a TACI protein and a first reportergene. The first reporter gene is controlled by an NF-AT promoter, andencodes a first marker protein. The TACI protein is activated, and theamount of expression of the first marker protein is quantified. Apotential drug is selected as a candidate drug when the amount of thefirst marker protein expressed in the presence of the candidate drug isdecreased relative to the amount expressed in the absence of thecandidate drug. The candidate drug is then contacted with a secondJurkat T cell that contains a T cell receptor and a second reportergene. The second reporter gene is controlled by an NF-AT promoter, andencodes a second marker protein. The T cell receptor is activated andthe amount of expression of the second marker protein is quantified andthen compared to the amount of second marker protein expressed in theabsence of the candidate drug. A candidate drug is identified as animmunosuppressant drug if either there is no decrease in the amount ofexpression of the second marker protein in the presence of the candidatedrug, or the decrease in the expression of the second marker protein ismeasurably less than the corresponding decrease in expression of thefirst marker protein in the presence of the candidate drug.

Any of the marker proteins described herein may be used for this aspectof the invention including SEAP, LacZ or luciferase. The first andsecond marker protein can be the same protein or two different proteins.The TACI protein may be activated with an antibody raised against a TACIprotein, or an active fragment thereof, or a fusion protein thereof. Ina preferred embodiment, the TACI protein is TACI-1. Several promoterscan be used to control the reporter gene including the NF-AT promotermentioned above and the AP-1 promoter. Potential drugs can be obtainedfrom any of the drug libraries currently available, and from thechemical and phage libraries described herein.

These and other aspects of the present invention will be betterappreciated by reference to the following drawings and DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The tissue distribution, protein sequence and other salientfeatures of TACI-1. FIG. 1 depicts a Northern blot of the tissuesindicated probed with TACI-1 cDNA. Other tissues probed include theheart, brain, placenta, lung, liver, skeletal muscle, kidney and thepancreas, none of which showed any TACI-1 mRNA expression.

FIG. 2A (SEQ ID NO:2) depicts the amino acid sequence of TACI-1. Theproposed transmembrane domain is shown in boxed print and the PrositeTNFR_NGFR motifs are underlined. FIG. 2B depicts a Kyte-Doolittlehydrophobicity plot and schematic diagram of TACI-1 showing thepositions of the putative transmembrane domain (solid black) and theTNFR_NGFR motifs (stippled). FIG. 2C depicts the cysteine residues ofthe TACI protein and other TNFR family members.

FIG. 3. TACI-1 is a cell surface protein. FIG. 3A depicts the flowcytometry of TAg Jurkat T cells transiently transfected with a TACI-1expression plasmid and the transfection marker, pHook (Invitrogen), orthe transfection marker pHook alone. Cell surface expression of TACI-1and the transfection marker protein, HA-Hook, were detected by indirectimmunofluorescence using the immunoaffinity-purified anti-TACI-1polyclonal antibody and the 12CA5 monoclonal antibody. Depicted datarepresent TACI-1 staining of cells gated for the transfection marker.FIG. 3B depicts a photomicrograph showing surface the staining of Cos-7cells transiently expressing N-terminal FLAG-tagged TACI-1, stained withM2 (anti-FLAG) antibodies and fluorescent anti-mouse IgG antibodies[Bram & Crabtree (1994) Nature 371:355-358]. Surface staining waspresent whether or not cells were permeabilised with detergent.

FIG. 4. TACI-1 is a signaling protein that functions in the activationof NF-AT-specific transcription. FIG. 4A depicts the activation of anNF-AT-driven secreted alkaline phosphatase reporter. TAg Jurkat T cells,co-transfected with the SXNFAT reporter [Bram et al. (1993) Mol. Cell.Biol. 13:4760-4769] and the expression plasmid pBJ5 [Takebe, Y., et al.(1988) Mol. Cell. Biol. 8:466-472] containing either TACI-1-encodingcDNA (solid triangles) or nothing (circles), were treated with 50 ng/mlPMA and the indicated amounts of ionomycin in the presence (closedsymbols) and absence (open symbols) of magnetic beads coated withimmunoaffinity-purified anti-TACI-1 polyclonal antibodies. FIG. 4Bdepicts the activation of NF-AT by antibody-cross linked TACI-1 that isblocked by Cyclosporin A (CsA) or FK506. NF-AT activation was determinedin TAg Jurkat T cells co-transfected with SXNFAT and pBJ5 (−TACI-1,left) or pBJ5-TACI-1 (+TACI-1, right), and treated with the indicatedcombinations of PMA (50 ng/ml), ionomycin (2 μM), CsA (100 ng/ml) andFK506 (500 pg/ml) in the presence of anti-TACI-1 antibody-coated beads(‘NS’, not stimulated). FIG. 4C shows that NF-AT activation byantibody-cross linked TACI-1 requires extracellular calcium. NF-ATactivation was measured in the presence of EGTA in Jurkat T cells overexpressing TACI-1. The treatments included cross linked anti-TACI-1(circles), transfection with a C-terminally truncated,calcium-independent calcineurin A subunit (triangles), or activationwith the TCR stimulating antibody OKT3 (squares). All cells wereco-stimulated by the addition of PMA to 50 ng/ml. FIG. 4D depicts theactivation of an AP-1-driven secreted alkaline phosphatase reporter.AP-1 activation was measured in TAg Jurkat T cells, co-transfected witha mouse metallothionein AP-1-SEAP reporter [Bram et al., 1991, supra]and pBJ5 containing no insert (−TACI-1, left) or TACI-1 cDNA (+TACI-1,right). Cells were incubated in the absence, and with seriallyincreasing amounts, of cross linked anti-TACI-1 antibodies. To controlfor transfection efficiency, a plasmid containing a constitutivepromoter driving the expression of luciferase (EF-Luc) was included.

FIG. 5. TACI-1 interaction with CAML is critical for Ca²⁺ signaling.FIG. 5A shows the yeast 2-hybrid interaction. Full-length cDNAs andindicated deletion mutants of TACI-1 and CAML were cloned into the yeastexpression plasmids pACT and/or pAS1, and the indicated combinationswere tested for interaction with the yeast 2-hybrid system (‘+’,positive interaction; ‘−’, no interaction; ‘ND’, not done). FIG. 5Bdepicts the co-immunoprecipitation of CAML with TACI-1. 293T cells weretransfected with the indicated combinations of the expression plasmidpBJ5 containing cDNAs for CAML, TACI-1 with an N-terminal FLAG tag, theN-terminal 146 amino acids of CAML (CLX91), or no insert. Afterincubation for 48 hours, the cells were lysed (1% dodecyl maltoside, 20mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 2 mM MgSO₄, 1 mM CaCl₂, 1 mMPMSF) and the lysate was clarified by centrifugation. FLAG-tagged TACI-1and associated proteins were immunoprecipitated with anti-FLAGmonoclonal antibody-conjugated agarose beads and subjected to Westerntransfer using standard protocols. The Western blot was probed withimmunoaffinity-purified anti-CAML polyclonal antibodies followed bychemiluminescent detection (Amersham). Parallel Western blots, performedfor each sample, confirmed the expected expression of TACI-1, CAML orthe truncated CAML mutant in all transfections. FIG. 5C shows that theover expression of the N-terminal half of CAML has a dominant negativeeffect on TACI-1-induced NF-AT activation. NF-AT activation wasdetermined in TAg Jurkat cells transfected with pBJ5 alone, pBJ5-TACI-1plus the control plasmid, or pBJ5-TACI-1 with an equivalent amount ofCLX91, following treatment with TACI-1-specific antibodies (top). TACI-1expression in these transfections was determined by Western blot usinganti-TACI-1 polyclonal antibodies (bottom). FIG. 5D depicts a schematicdiagram showing the TACI-1/CAML signal transduction model (‘SOC’,stores-operated calcium channel).

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in its broadest aspect, provides a novel cellsurface receptor that is normally present in B-lymphocytes, and to amuch lesser extent in immature T-lymphocytes. The role of the cellsurface receptor, the Transmembrane Activator and CAML-Interactor (TACI)protein, is to participate in alternate or co-stimulatory pathways toactivate or control lymphocyte function. These functions can includeresponse of lymphocytes to foreign antigens in infection, or to cancer,in the graft-rejection, and graft-vs-host reaction. Additionally,activation of lymphocyte signaling plays a key role during lymphocytedevelopment, thus allowing the positive selection of functionallymphocytes and negative selection against self-reactive clones. Whenactivated, the TACI protein stimulates the influx of calcium inlymphocytes. Such calcium influx can, under specific circumstances, leadto the onset of programmed cell death (apoptosis).

The terms “Transmembrane Activator and CAML-Interactor” protein or“TACI” or “TACI protein” are used herein interchangeably with“transmembrane CAML-binding protein” or “TCB” or “TCB protein” and referto proteinaceous material including single or multiple proteins that actas a novel cell surface receptor that is normally present inB-lymphocytes. This cell surface receptor has the profile of activitiesset forth herein. Accordingly, proteins displaying substantiallyequivalent or altered activity are likewise contemplated. Thesemodifications may be deliberate, for example, such as modificationsobtained through site-directed mutagenesis, or may be accidental, suchas those obtained through mutations in hosts that are producers of theprotein. Included within the scope of these terms are proteinsspecifically recited herein, as well as all substantially homologousanalogs and allelic variations.

In one embodiment the Transmembrane Activator and CAML-Interactor isTACI-1, the human homologue. As shown in the Example, infra, TACI-1initiates a previously undetected signal transduction mechanism thatdirectly links cell surface stimuli to the intracellular signalingmolecule, CAML. TACI-1 therefore is a member of a new class oflymphocyte-specific cell surface receptors that modulate the immuneresponse and thus may be used as tool to regulate the immune system ineither a positive or a negative direction. In a specific embodiment,TACI-1 has the amino acid sequence set forth in SEQ ID NO:2.

As used herein when a particular nucleic acid is said to encode aprotein or polypeptide of the present invention, it is meant that theportion of the particular nucleic acid that consists of the codingsequence for that protein or polypeptide is being identified. Forexample, the coding region of SEQ ID NO:1 is from nucleotide 14 tonucleotide 895, including the 3 nucleotide translation stop codon at the3/terminus.

For many purposes, there is a substantial interest in being able toselectively prevent activation of lymphocytes or in the alternative toselectively enhance their activation. For example, for lymphocytemediated autoimmune diseases, transplant rejection syndrome, andgraft-versus host disease, inhibiting the activation of the lymphocyteinvolved is a viable or necessary treatment for the disease.Furthermore, in the case of myelomas, lymphomas, and leukemias,especially of B cells or immature T cells, there is an interest inslowing the proliferation of cancer cells, which may allow for therapieswhich are not as destructive to the host as present day therapies. Onthe other hand, for infections or anti-tumor immune responses, therewould be interest in being able to activate lymphocytes to more rapidlyrespond to the pathogen. Thus, the present invention enables selectionof useful agents, e.g., synthetic organic compounds which can activateand/or deactivate lymphocytes by providing a previously unknown keycomponent in the lymphocyte activation pathway.

In addition, as one understands the activation pathway more completely,one is able to modulate the pathway more effectively, such as providingfor agents which are selective for a particular set or subset of acellular population. Since in many cases activation requiresco-stimulation, being able to manipulate agents available to the cellmay allow for such cellular activity. In this context, theidentification of a target protein that can be used to develop drugsthat modulate a particular pathway, such as the CAML-mediated activationpathway, would allow physicians to particularly treat distinct immuneconditions without over-stimulating or over-suppressing other delicateaspects of the immune system that are otherwise functioning well. Suchtargeted stimulation can be used to specifically amplify the effects ofimmune stimulators, such as IL-2, thus allowing for the use of lowerdoses of the immune stimulator and reducing side effects.

Furthermore, the invention is an important advance in understanding theCAML-mediated activation pathway, by permitting selective evaluation andcontrol over the presence or the absence of a particular intermediate inthat pathway. This can be achieved with a knock-out animal usinghomologous recombination, integration of genes providing for antisensesequences, introduction of expression constructs involving induciblepromoters, and the like. There is also an interest in being able todetermine when a particular gene is being expressed or is silent, thenature of the cells in which the protein is expressed, and the like.Therefore, there is substantial interest in identifying specificcomponents of cellular pathways to allow for understanding an activationpathway, selectively modulating that pathway, and developing drugs whichmay be active in binding to the target protein. In this way, drugs canbe screened to inhibit such specific pathways.

One particular aspect of the present invention includes a drug screenthat uses TACI as a tool for developing immunosuppressant drugs specificfor B-lymphocytes. Such immunosuppressant drugs would selectively blockthe action of B-lymphocytes, while leaving T-cells intact to protectpatients from viral pathogens. These drugs would be useful in treatingdiseases such as Systemic Lupus Erythematosus, a disease due to anover-activation of the B-lymphocyte response, or multiple myeloma (e.g.,Bence-Jones Myeloma).

Cross-linking the TACI protein activates calcium influx and potentiallyother secondary messengers. Its mode of action can be mediated by CAML.Unlike known cell surface signaling molecules that are specific for Bcells or T cells, such as CD4, CD8, TCR, and CD3, the novel cell surfacereceptor of the present invention physically interacts with CAML. Inaddition, there is no sequence homology between the novel cell surfacereceptor of the present invention and any of these known lymphocyte cellsurface signaling molecules, or any other cell surface signalingmolecules.

TACI Proteins and Polypeptides

In a broad embodiment, the present invention provides TACI proteins.Such proteins include an extracellular domain, a transmembrane domain,and a cytoplasmic domain. The extracellular domain binds ligand. Uponligand binding, the cytoplasmic domain binds CAML, thus initiating aCa²⁺-dependent activation pathway. Receptor oligomerization, e.g., bybinding ligand or with an anti-receptor antibody, also initiates anon-Ca²⁺-dependent activation pathway.

The monomeric form of a TACI protein contains about 295 amino acids. Asused herein “about 295 amino acids” means between 265 to 325 aminoacids, i.e., roughly plus or minus 10%.

The invention further relates to functionally active polypeptidecomponents of TACI. In one aspect, a functionally active component ofTACI is an antigenic fragment, e.g., a peptide reactive with anti-TACIantibodies, or which, when conjugated to a carrier, can be used togenerate anti-TACI antibodies. Another functionally active fragmentincludes the extracellular domain, which binds ligand. The extracellulardomain corresponds to the N-terminal fragment of TACI, e.g., from thefirst amino acid residue of mature TACI to the transmembrane domain. Ina specific embodiment, the extracellular domain has the amino acidsequence corresponding to about residue 1 to about residue 166 of SEQ IDNO:6. The ligand-binding region of TACI is a sub-fragment of theN-terminal fragment corresponding to the extracellular domain.

Still another functionally active fragment is the cytoplasmic domain,e.g., from the C-terminal end of the transmembrane domain to theC-terminus of TACI. The cytoplasmic domain of TACI mediates signaltransduction via Ca⁺-dependent and Ca²⁺-independent mechanisms. Thecytoplasmic domain includes the CAML-binding region of TACI. Inparticular, this domain binds a polypeptide corresponding to theN-terminal 146 amino acid residues of CAML. In a specific embodiment,the cytoplasmic domain corresponds to from about amino acid residue 187to about amino acid residue 293 of SEQ ID NO:2.

In yet another embodiment, the present invention provides proteolyticfragments of a TACI protein. Such fragments can be prepared by enzymaticdigestion, e.g., with Saureus Polypeptides V8 in papain trypsin,chymotrypsin, cathepsin, collagenase, enteropeptidase, thrombin, orfibrinolytic or clotting enzymes; by chemical cleavage, e.g., withcyanogen bromide, or sodium borohydride; etc.

In a specific embodiment, the TACI protein is a receptor protein havingthe amino acid sequence as shown in SEQ ID NO:2, from residue 1 toresidue 293. The present invention contemplates allelic variants ofTACI, homologous TACI proteins from other species, and TACI analogs,e.g., prepared by making conservative amino acid substitutions, whetherby genetic engineering or by chemical synthesis. A TACI analogue of theinvention also includes TACI antigenic fragments that contain, e.g, aterminal cysteine residue to facilitate cross-linking to a carrierprotein.

There are no reported DNA sequences that are closely related to that forTACI-1. A search for Prosite motifs in TACI-1 reveals one TNFR_NGFRpattern, which consists ofC-x(4,6)-[FYH]-x(5,10)—C-x(0,2)—C-x(2,3)—C-x(7,11)—C-x(4,6)-[DNEQSKP]-x(2)-C(SEQ ID NO:11) in the N-terminal half of the protein (where e.g.,C-x(4,6)-[FYH] is indicative of an amino acid sequence starting withcysteine followed by either 4, 5, or 6 unspecified amino acids, furtherfollowed by either a phenylalanine, a tyrosine, or a histidine). Thismotif is found in a number of proteins, most of which are receptors forgrowth factors. Some of these proteins have one copy of this motif. Acomparison of the TACI-1 protein sequence with itself reveals asignificant repeat between the TNFR_NGFR motif at residues 33-66 andresidues 70-104. This analysis drew attention to the presence of twoTNFR-type cysteine-rich domains encompassing these regions that indicatethat TACI-1 is a member of the superfamily of TNFR receptors.

Synthetic TACI Polypeptides.

The term “polypeptide” is used in its broadest sense to refer to acompound of two or more subunit amino acids, amino acid analogs, orpeptidomimetics. The subunits may be linked by peptide bonds. In anotherembodiment, the subunit may be linked by other bonds, e.g., ester,ether, etc. As used herein the term “amino acid” refers to eithernatural and or unnatural or synthetic amino acids, including glycine andboth the D or L optical isomers, and amino acid analogs andpeptidomimetics. A peptide of three or more amino acids is commonlycalled an oligopeptide if the peptide chain is short. If the peptidechain is long, the peptide is commonly called a polypeptide or aprotein. According to the invention, TACI fragments, such as antigenicfragments, or potentially even full-length TACI, can be preparedsynthetically.

Synthetic polypeptides, prepared using the well known techniques ofsolid phase, liquid phase, or peptide condensation techniques, or anycombination thereof, can include natural and unnatural amino acids.Amino acids used for peptide synthesis may be standard Boc (N^(α)-aminoprotected Nα-t-butyloxycarbonyl) amino acid resin with the standardde-protecting, neutralization, coupling and wash protocols of theoriginal solid phase procedure of Merrifield [(1963) J. Am. Chem. Soc.85:2149-2154], or the base-labile N^(α)-amino protected9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpinoand Han [(1972) J. Org. Chem. 37:3403-3409]. Both Fmoc and BocN^(α)-amino protected amino acids can be obtained from Fluka, Bachem,Advanced Chemtech, Sigma, Cambridge Research Biochemical, Bachem, orPeninsula Labs or other chemical companies familiar to those whopractice this art. In addition, the method of the invention can be usedwith other N^(α)-protecting groups that are familiar to those skilled inthis art. Solid phase peptide synthesis may be accomplished bytechniques familiar to those in the art and provided, for example, inStewart and Young (1984) Solid Phase Synthesis (2d Ed., Pierce ChemicalCo., Rockford, Ill.); Fields and Noble (1990) Int. J. Pept. Protein Res.35:161-214, or using automated synthesizers, such as sold by ABS. Thus,polypeptides of the invention may comprise D-amino acids, a combinationof D- and L-amino acids, and various “designer” amino acids (e.g.,β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids,etc.) to convey special properties. Synthetic amino acids includeornithine for lysine, fluorophenylalanine for phenylalanine, andnorleucine for leucine or isoleucine. Additionally, by assigningspecific amino acids at specific coupling steps, α-helices, β turns, βsheets, γ-turns, and cyclic peptides can be generated.

In a further embodiment, subunits of peptides that confer usefulchemical and structural properties will be chosen. For example, peptidescomprising D-amino acids will be resistant to L-amino acid-specificproteases in vivo. In addition, the present invention envisionspreparing peptides that have more well defined structural properties,and the use of peptidomimetics, and peptidomimetic bonds, such as esterbonds, to prepare peptides with novel properties. In another embodiment,a peptide may be generated that incorporates a reduced peptide bond,i.e., R₁—CH₂—NH—R₂, where R₁ and R₂ are amino acid residues orsequences. A reduced peptide bond may be introduced as a dipeptidesubunit. Such a molecule would be resistant to peptide bond hydrolysis,e.g., protease activity. Such peptides would provide ligands with uniquefunction and activity, such as extended half-lives in vivo due toresistance to metabolic breakdown, or protease activity. Furthermore, itis well known that in certain systems constrained peptides show enhancedfunctional activity [Hruby (1982) Life Sciences 31:189-199]; [Hruby etal. (1990) Biochem J. 268:249-262]; the present invention provides amethod to produce a constrained peptide that incorporates randomsequences at all other positions.

Non-Classical Amino Acids that Induce Conformational Constraints:

The following non-classical amino acids may be incorporated in thepeptide in order to introduce particular conformational motifs:1,2,3,4-tetrahydroisoquinoline-3-carboxylate [Kazmierski et al. (1991)J. Am. Chem. Soc. 113:2275-2283]; (2S,3S)-methyl-phenylalanine,(2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and(2R,3R)-methyl-phenylalanine [Kazmierski and Hruby (1991) TetrahedronLett.]; 2-aminotetrahydronaphthalene-2-carboxylic acid [Landis, Ph.D.Thesis, University of Arizona, (1989)];hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate [Miyake et al.(1989) J. Takeda Res. Labs. 43:53-76]; β-carboline (D and L)[Kazmierski, Ph.D. Thesis, University of Arizona, (1988)]; HIC(histidine isoquinoline carboxylic acid) [Zechel et al. (1991) Int. J.Pep. Protein Res. 43]; and HIC (histidine cyclic urea) (Dharanipragada).

The following amino acid analogs and peptidomimetics may be incorporatedinto a peptide to induce or favor specific secondary structures: LL-Acp(LL-3-amino-2-propenidone-6-carboxylic acid), a β-turn inducingdipeptide analog (Kemp et al. (1985) J. Org. Chem. 50:5834-5838];β-sheet inducing analogs [Kemp et al. (1988) Tetrahedron Lett.29:5081-5082]; β-turn inducing analogs [Kemp et al. (1988) TetrahedronLett. 29:5057-5060]; α-helix inducing analogs [Kemp et al. (1988)Tetrahedron Lett. 29:4935-4938]; γ-turn inducing analogs [Kemp et al.(1989) J. Org. Chem. 54:109:115]; and analogs provided by the followingreferences: Nagai and Sato (1985) Tetrahedron Lett. 26:647-650; DiMaioet al. (1989) J. Chem. Soc. Perkin Trans., p. 1687; also a Gly-Ala turnanalog [Kahn et al. (1989) Tetrahedron Lett. 30:2317]; amide bondisostere [Jones et al. (1988) Tetrahedron Lett. 29:3853-3856]; tretrazol[Zabrocki et al. (1988) J. Am. Chem. Soc. 110:5875-5880]; DTC [Samanenet al. (1990) Int. J. Protein Pep. Res. 35:501:509]; and analogs taughtin Olson et al. (1990) J. Am. Chem. Sci. 112:323-333 and Garvey et al.(1990) J. Org. Chem. 56:436. Conformationally restricted mimetics ofbeta turns and beta bulges, and peptides containing them, are describedin U.S. Pat. No. 5,440,013, issued Aug. 8, 1995 to Kahn.

Protein Derivatives.

There are two major classes of peptide-carbohydrate linkages toproteins. First, ether bonds join the serine or threonine hydroxyl to ahydroxyl of the sugar. Second, amide bonds join glutamate or aspartatecarboxyl groups to an amino group on the sugar. Acetal and ketal bondsmay also bind carbohydrate to peptide.

Generally, the TACI protein, or a fragment thereof, such as theN-terminal extracellular domain fragment or the C-terminal cytoplasmicCAML-binding domain fragment, may be derivatized by the attachment ofone or more chemical moieties to the protein moiety. Chemicalmodification of biologically active component or components may provideadditional advantages under certain circumstances, such as increasingthe stability and circulation time of the component or components anddecreasing immunogenicity. See U.S. Pat. No. 4,179,337, Davis et al.,issued Dec. 18, 1979. For a review, see Abuchowski et al., in Enzymes asDrugs [J. S. Holcerberg and J. Roberts, eds. (1981) pp. 367-383]. Areview article describing protein modification and fusion proteins isFrancis (1992) Focus on Growth Factors 3:4-10, Mediscript: MountviewCourt, Friern Barnet Lane, London N20, OLD, UK.

The chemical moieties suitable for derivatization may be selected fromamong water soluble and water insoluble polymers, with water solublepolymers preferred. The polymer selected should preferably be watersoluble so that the component to which it is attached does notprecipitate in an aqueous environment, such as a physiologicalenvironment. Preferably, for therapeutic use of the end-productpreparation, the polymer will be pharmaceutically acceptable. Oneskilled in the art will be able to select the desired polymer based onsuch considerations as whether the polymer/component conjugate will beused therapeutically, and if so, the desired dosage, circulation time,resistance to proteolysis, and other considerations. For the presentcomponent or components, these may be ascertained using the assaysprovided herein.

Labeled TACI Proteins.

The TACI protein of the present invention, and fragments thereof, may belabeled. Suitable labels include enzymes, fluorophores (e.g.,fluorescence isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR),rhodamine, free or chelated lanthanide series salts, especially Eu³⁺, toname a few fluorophores), chromophores, radioisotopes, chelating agents,dyes, colloidal gold, latex particles, ligands (e.g., biotin), andchemiluminescent agents. When a control marker is employed, the same ordifferent labels may be used for the test sample and control marker.

In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re areused, known currently available counting procedures may be utilized. Inthe instance where the label is an enzyme, detection may be accomplishedby any of the presently utilized calorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques known inthe art.

Direct labels are one example of labels which can be used according tothe present invention. A direct label has been defined as an entity,which in its natural state, is readily visible, either to the naked eye,or with the aid of an optical filter and/or applied stimulation, e.g.U.V. light to promote fluorescence. Among examples of colored labels,which can be used according to the present invention, include metallicsol particles, for example, gold sol particles such as those describedby Leuvering (U.S. Pat. No. 4,313,734); dye sole particles such asdescribed by Gribnau et al. (U.S. Pat. No. 4,373,932) and May et al. (WO88/08534); dyed latex such as described by May, supra, Snyder (EP-A 0280 559 and 0 281 327); or dyes encapsulated in liposomes as describedby Campbell et al. (U.S. Pat. No. 4,703,017). Other direct labelsinclude a radio nucleotide, a fluorescent moiety or a luminescentmoiety. In addition to these direct labeling devices, indirect labelscomprising enzymes can also be used according to the present invention.Various types of enzyme linked immunoassays are well known in the art,for example, alkaline phosphatase and horseradish peroxidase, lysozyme,glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease, theseand others have been discussed in detail by Eva Engvall in EnzymeImmunoassay ELISA and EMIT in Methods in Enzymology (1980) 70:419-439and in U.S. Pat. No. 4,857,453.

Suitable enzymes include, but are not limited to, alkaline phosphataseand horseradish peroxidase.

Other labels for use in the invention include magnetic beads or magneticresonance imaging labels.

In another embodiment, a phosphorylation site can be created on anantibody of the invention for labeling with ³²P, e.g., as described inEuropean Patent No. 0372707 (application No. 89311108.8) by Pestka, orU.S. Pat. No. 5,459,240, issued Oct. 17, 1995 to Foxwell et al.

As exemplified herein, proteins, including antibodies, can be labeled bymetabolic labeling. Metabolic labeling occurs during in vitro incubationof the cells that express the protein in the presence of culture mediumsupplemented with a metabolic label, such as [³⁵S]-methionine or[³²P]-orthophosphate. In addition to metabolic (or biosynthetic)labeling with [³⁵S]-methionine, the invention further contemplateslabeling with [¹⁴C]-amino acids and [³H]-amino acids (with the tritiumsubstituted at non-labile positions).

Chimeric TACI Proteins

A chimeric TACI protein of the invention may be a protein that isgenerated by joining a functional domain of a TACI protein, such as theligand binding domain or the CAML-binding domain, with the complementarydomain of another protein, e.g., an alternative receptor. Chimericconstructs can also be prepared with a functionally active fragment of aTACI protein and another functionally active molecule. For example, theextracellular domain of a TACI protein may be joined to the Fc domain ofan immunoglobulin. Alternatively, the cytoplasmic domain of a TACIprotein could be joined to a receptor ligand, such as transferrin or ahormone, for intracellular targeting. In yet another embodiment, a TACIdomain could be joined to another targeting molecule, such as ananti-immunoglobulin heavy chain or light chain molecule (e.g, an Fvportion of an antibody) to specifically target B cells. In still anotherembodiment, the functionally active fragment of a TACI protein,preferably the N-terminal extracellular domain, can be joined with aglycosylphospholipid, such as glycosylphosphoinositol, anchor signalsequence, preferably located at the C-terminus of the TACI fragment, sothat a glycolipid anchored protein is generated [Cross (1990) Annu. Rev.Cell Biol. 6: 1-39; Low (1987) Biochem. J. 244:1-13].

A chimeric TACI receptor can be prepared by joining the extracellulardomain of another receptor molecule with a transmembrane domain and theintracellular domain of a TACI protein. In another embodiment, theextracellular domain of TACI can be joined with a transmembrane domainand an intracellular domain of another receptor molecule. Thetransmembrane domain can be the transmembrane domain of a TACI protein,the transmembrane domain of the other receptor, or a differenttransmembrane domain. Preferably, the transmembrane domain is from thesame protein component of the chimera as the extracellular domain.Chimeric receptors have been described [International PatentPublications WO96/23814; WO96/23881; and WO96/24671]. Chimeric antigenreceptors have been described [Capon et al., U.S. Pat. No. 5,359,046,issued Oct. 25, 1994], including functional antigen-specific receptorsgenerated in B cells [Sanchez et al. (1993) J. Exp. Med. 178:1049-1055]and T cells [Burkhardt et al. (1994) Mol. Cell. Biol. 14:1095-1103] byfusing the Igα and Igβ signal transduction chains to IgM. Various type Iand type II cytokine receptors, including interferon-α, interferon-β,interferon-γ, interleukin-1, interleukin-2, interleukin-3,interleukin-4, interleukin-6, interleukin-8, interleukin-10,interleukin-12, erythropoietin, granulocyte-macrophage colonystimulating factor (CSF), granulocyte-CSF, macrophage-CSF, α-chemokinereceptors, and β-chemokine receptors, can provide complementarycomponents in a chimeric receptor comprising a functionally activefragment of a TACI protein.

As those of ordinary skill in the art can appreciate, transmembranedomains are generally functionally equivalent for anchoring a protein ina membrane. However, the presence of specific amino acid residues in atransmembrane domain can affect receptor interaction with, e.g.,dimerization, or association with other integral membrane proteins, suchas in a multi-protein receptor complex. Thus, selection of atransmembrane domain depends on whether regulatory functions performedby the transmembrane domain are desired or necessary.

Nucleic Acids Encoding TACI Proteins

The present invention contemplates isolation of a gene encoding a TACIprotein including a full length, or naturally occurring form of TACIprotein, and any antigenic fragments thereof from any animal,particularly mammalian and more particularly human, source. As usedherein, the term “gene” refers to an assembly of nucleotides that encodea polypeptide, and includes cDNA and genomic DNA nucleic acids.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis (1989)Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook etal., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N.Glover ed. (1985)); Oligonucleotide Synthesis (M. J. Gait ed. (1984));Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)];Transcription And Translation [B. D. Hames & S. J. Higgins, eds.(1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; ImmobilizedCells And Enzymes [IRL Press, (1986)]; B. Perbal (1984) A PracticalGuide To Molecular Cloning; F. M. Ausubel et al. (eds.) (1994) CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc.

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “replicon” is any genetic element (e.g.,plasmid, chromosome, virus) that functions as an autonomous unit of DNAreplication in vivo, i.e., capable of replication under its own control.

A “cassette” refers to a segment of DNA that can be inserted into avector at specific restriction sites. The segment of DNA encodes apolypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

A cell has been “transfected” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. A cell has been “transformed”by exogenous or heterologous DNA when the transfected DNA effects aphenotypic change. Preferably, the transforming DNA should be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m) of55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide;or 30% formamide, 5×SSC, 0.5% SDS. Moderate stringency hybridizationconditions correspond to a higher T_(m), e.g., 40% formamide, with 5× or6×SCC. High stringency hybridization conditions correspond to thehighest T_(m), e.g., 50% formamide, 5× or 6×SCC. Hybridization requiresthat the two nucleic acids contain complementary sequences, althoughdepending on the stringency of the hybridization, mismatches betweenbases are possible. The appropriate stringency for hybridizing nucleicacids depends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the greaterthe value of T_(m) for hybrids of nucleic acids having those sequences.The relative stability (corresponding to higher T_(m)) of nucleic acidhybridizations decreases in the following order: RNA:RNA, DNA:RNA,DNA:DNA. For hybrids of greater than 100 nucleotides in length,equations for calculating T_(m) have been derived (see Sambrook et al.,supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook et al., supra, 11.7-11.8). A minimum length for a hybridizablenucleic acid is at least 10 nucleotides; preferably at least 18nucleotides; more preferably the length is at least 24 nucleotides andmost preferably at least 30 nucleotides in length. In a specificembodiment, a hybridizable nucleic acid of the invention has a sequencecorresponding to at least 12 nucleotides, preferably at least 18nucleotides, more preferably at least 24 nucleotides, and mostpreferably at least 30 nucleotides in length of SEQ ID NO:1, or morespecifically the coding sequence of SEQ ID NO:1.

In a specific embodiment, the term “standard hybridization conditions”refers to a T_(m) of 55° C., and utilizes conditions as set forth above.In a preferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 65° C.

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of at least 18 nucleotides, that is hybridizable to a genomicDNA molecule, a cDNA molecule, or an mRNA molecule encoding a TACIprotein. Oligonucleotides can be labeled, e.g., with ³²P-nucleotides ornucleotides to which a label, such as biotin, has been covalentlyconjugated (see the discussion, supra, with respect to labeling TACIpolypeptides). In one embodiment, a labeled oligonucleotide can be usedas a probe to detect the presence of a nucleic acid encoding a TACIprotein. In another embodiment, oligonucleotides (one or both of whichmay be labeled) can be used as PCR primers, either for cloning fulllength or a fragment of a TACI protein, or to detect the presence ofnucleic acids encoding a TACI protein. In a further embodiment, anoligonucleotide of the invention can form a triple helix with a TACI DNAmolecule. Generally, oligonucleotides are prepared synthetically,preferably on a nucleic acid synthesizer. Accordingly, oligonucleotidescan be prepared with non-naturally occurring phosphoester analog bonds,such as thioester bonds, etc.

“Homologous recombination” refers to the insertion of a foreign DNAsequence of a vector in a chromosome. Preferably, the vector targets aspecific chromosomal site for homologous recombination. For specifichomologous recombination, the vector will contain sufficiently longregions of homology to sequences of the chromosome to allowcomplementary binding and incorporation of the vector into thechromosome. Longer regions of homology, and greater degrees of sequencesimilarity, may increase the efficiency of homologous recombination.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′(carboxyl) terminus. A coding sequence can include, but is not limitedto, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence. In aspecific embodiment, a TACI coding sequence of the invention has thenucleotide sequence depicted in SEQ ID NO:1.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced and translated into the protein encoded by the coding sequence.

A “signal sequence” may be included at the beginning of the codingsequence of a protein to be expressed on the surface of a cell. Thissequence encodes a signal peptide, N-terminal to the mature polypeptide,that directs the host cell to translocate the polypeptide. The term“translocation signal sequence” is used herein to refer to this sort ofsignal sequence. Translocation signal sequences can be found associatedwith a variety of proteins native to eukaryotes and prokaryotes, and areoften functional in both types of organisms. Interestingly, the TACI ofthe invention is transported so that the N-terminus is extracellular inthe absence of a cleaved signal sequence. Thus, this transmembraneprotein is a type III transmembrane protein [see Wilson-Rawls et al.(1994) Virology 201:66-76]. Thus, in a construct of the presentinvention (including a chimeric construct as discussed above), if theN-terminal portion of the construct encodes the N-terminus of TACI, asignal peptide may not be required to obtain expression of thetransmembrane TACI protein.

As used herein, the term “sequence homology” in all its grammaticalforms refers to the relationship between proteins that possess a “commonevolutionary origin,” including proteins from superfamilies (e.g., theimmunoglobulin superfamily) and homologous proteins from differentspecies (e.g., myosin light chain, etc.) [Reeck et al. (1987) Cell50:667]. The present invention naturally contemplates homologues of thehuman TACI protein as falling within the scope of the invention.

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences whether or not they share a common evolutionaryorigin (see Reeck et al., supra). In common usage, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and not a common evolutionary origin.

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%(preferably at least about 75%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra. The present inventioncontemplates nucleotides that are 50% similar to SEQ ID NO:1 (or itscomplementary sequence), preferably 60% similar, and more preferably 75%similar. Preferably such substantially similar nucleic acids arehomologous.

Similarly, in a particular embodiment, two amino acid sequences are“substantially homologous” or “substantially similar” when greater than30% of the amino acids are identical, or greater than about 60% aresimilar (functionally identical). Preferably, the similar or homologoussequences are identified by alignment using, for example, the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program.

The term “corresponding to” is used herein to refer similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

A gene encoding a TACI protein, whether genomic DNA or cDNA, can beisolated from any source, particularly from a human cDNA or genomiclibrary. Methods for obtaining TACI protein gene are well known in theart, as described above (see, e.g., Sambrook et al., 1989, supra).

Accordingly, any animal cell potentially can serve as the nucleic acidsource for the molecular cloning of a TACI protein gene. The DNA may beobtained by standard procedures known in the art from cloned DNA (e.g.,a DNA “library”), and preferably is obtained from a cDNA libraryprepared from tissues with high level expression of the protein (e.g., athymic cDNA library, since peripheral blood cells and in particularlymphocyte cells, appear to have the highest levels of expression ofTACI protein), by chemical synthesis, by cDNA cloning, or by the cloningof genomic DNA, or fragments thereof, purified from the desired cell(see, for example, Sambrook et al., 1989, supra; Glover, D. M. (ed.)(1985) DNA Cloning. A Practical Approach, MRL Press, Ltd., Oxford, U.K.Vol. I, II). Clones derived from genomic DNA may contain regulatory andintron DNA regions in addition to coding regions; clones derived fromcDNA will not contain intron sequences. Whatever the source, the geneshould be molecularly cloned into a suitable vector for propagation ofthe gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired TACI protein gene may be accomplished ina number of ways. For example, a portion of a TACI protein gene or itsspecific RNA, or a fragment thereof, can be purified and labeled, thegenerated DNA fragments may then be screened by nucleic acidhybridization to the labeled probe [Benton and Davis (1977) Science196:180]; [Grunstein and Hogness (1975) Proc. Natl. Acad. Sci. U.S.A.,72:3961]. For example, a set of oligonucleotides corresponding to thepartial amino acid sequence information obtained for the TACI proteincan be prepared and used as probes for DNA encoding a TACI protein, oras primers for cDNA or mRNA (e.g., in combination with a poly-T primerfor RT-PCR). Preferably, a fragment is selected that is highly unique tothe TACI protein of the invention. Those DNA fragments with substantialhomology to the probe will hybridize. As noted above, the greater thedegree of homology, the more stringent hybridization conditions can beused. In a specific embodiment, stringency hybridization conditions areused to identify a homologous TACI protein gene.

Further selection can be carried out on the basis of the properties ofthe gene, e.g., if the gene encodes a protein product having theisoelectric, electrophoretic, amino acid composition, or partial aminoacid sequence of the TACI protein as disclosed herein. Thus, thepresence of the gene may be detected by assays based on the physical,chemical, or immunological properties of its expressed product. Forexample, cDNA clones, or DNA clones which hybrid-select the propermRNAs, can be selected which produce a protein that, e.g., has similaror identical electrophoretic migration, isoelectric focusing ornon-equilibrium pH gel electrophoresis behavior, proteolytic digestionmaps, or antigenic properties as known for TACI protein.

A TACI protein gene of the invention can also be identified by mRNAselection, i.e., by nucleic acid hybridization followed by in vitrotranslation. In this procedure, nucleotide fragments are used to isolatecomplementary mRNAs by hybridization. Such DNA fragments may representavailable, purified TACI protein DNA, or may be syntheticoligonucleotides designed from the partial amino acid sequenceinformation. Immunoprecipitation analysis or functional assays (e.g.,CAML binding activity) of the in vitro translation products of theproducts of the isolated mRNAs identifies the mRNA and, therefore, thecomplementary DNA fragments, that contain the desired sequences. Inaddition, specific mRNAs may be selected by adsorption of polysomesisolated from cells to immobilized antibodies specifically directedagainst TACI protein, such as the rabbit polyclonal anti-human TACIprotein antibody described herein.

A radiolabeled TACI protein cDNA can be synthesized using the selectedmRNA (from the adsorbed polysomes) as a template. The radiolabeled mRNAor cDNA may then be used as a probe to identify homologous TACI proteinDNA fragments from among other genomic DNA fragments.

The present invention also relates to cloning vectors containing genesencoding analogs and derivatives of the TACI protein of the invention,that have the same or homologous functional activity as TACI protein,and homologs thereof from other species. The production and use ofderivatives and analogs related to TACI protein are within the scope ofthe present invention. In a specific embodiment, the derivative oranalog is functionally active, i.e., capable of exhibiting one or morefunctional activities associated with a full-length, wild-type TACIprotein of the invention. In another embodiment, TACI protein containinga different cytoplasmic domain, e.g., one unable to bind CAML but stillable to modulate the activation of AP-1. In another aspect, a TACIprotein of the invention can be prepared with a lectin domain or domainsfrom another protein, such as the mannose receptor of macrophages or thephospholipase receptor on muscle.

TACI protein derivatives can be made by altering encoding nucleic acidsequences by substitutions, additions or deletions that provide forfunctionally equivalent molecules. Preferably, derivatives are made thathave enhanced or increased functional activity relative to native TACIprotein. Alternatively, such derivatives may encode soluble fragments ofTACI protein extracellular domain that have the same or greater affinityfor the natural ligand of TACI protein of the invention. Such solublederivatives may be potent inhibitors of ligand binding to TACI protein.

Due to the degeneracy of nucleotide coding sequences, other DNAsequences which encode substantially the same amino acid sequence as aTACI protein gene may be used in the practice of the present invention.These include but are not limited to allelic genes, homologous genesfrom other species, and nucleotide sequences comprising all or portionsof TACI protein genes which are altered by the substitution of differentcodons that encode the same amino acid residue within the sequence, thusproducing a silent change. Likewise, the TACI protein derivatives of theinvention include, but are not limited to, those containing, as aprimary amino acid sequence, all or part of the amino acid sequence of aTACI protein including altered sequences in which functionallyequivalent amino acid residues are substituted for residues within thesequence resulting in a conservative amino acid substitution. And thus,such a substitution is defined as a conservative substitution.

For example, one or more amino acid residues within the sequence can besubstituted by another amino acid of a similar polarity, which acts as afunctional equivalent, resulting in a silent alteration. Substitutes foran amino acid within the sequence may be selected from other members ofthe class to which the amino acid belongs. For example, the non-polar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. Amino acidscontaining aromatic ring structures are phenylalanine, tryptophan, andtyrosine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Such alterations will not be expected to affect apparentmolecular weight as determined by polyacrylamide gel electrophoresis, orisoelectric point. Particularly preferred substitutions are:

Lys for Arg and vice versa such that a positive charge may bemaintained;

Glu for Asp and vice versa such that a negative charge may bemaintained;

Ser for Thr such that a free —OH can be maintained; and

Gln for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced at a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces β-turns in the protein's structure.

The genes encoding TACI protein derivatives and analogs of the inventioncan be produced by various methods known in the art. The manipulationswhich result in their production can occur at the gene or protein level.For example, the cloned TACI protein gene sequence can be modified byany of numerous strategies known in the art (Sambrook et al., 1989,supra). The sequence can be cleaved at appropriate sites withrestriction endonuclease(s), followed by further enzymatic modificationif desired, isolated, and ligated in vitro. In the production of thegene encoding a derivative or analog of TACI protein, care should betaken to ensure that the modified gene remains within the sametranslational reading frame as the TACI protein gene, uninterrupted bytranslational stop signals, in the gene region where the desiredactivity is encoded.

Additionally, the TACI protein-encoding nucleic acid sequence can bemutated in vitro or in vivo, to create and/or destroy translation,initiation, and/or termination sequences, or to create variations incoding regions and/or form new restriction endonuclease sites or destroypreexisting ones, to facilitate further in vitro modification.Preferably, such mutations enhance the functional activity of themutated TACI protein gene product. Any technique for mutagenesis knownin the art can be used, including but not limited to, in vitrosite-directed mutagenesis [Hutchinson et al. (1978) J. Biol. Chem.253:6551]; [Zoller and Smith (1984) DNA 3:479-488]; [Oliphant et al.(1986) Gene 44:177]; [Hutchinson et al. (1986) Proc. Natl. Acad. Sci.U.S.A. 83:710], use of TAB® linkers (Pharmacia), etc. PCR techniques arepreferred for site directed mutagenesis (see Higuchi, “Using PCR toEngineer DNA”, in PCR Technology: Principles and Applications for DNAAmplification, H. Erlich, ed. (1989) Stockton Press, Chapter 6, pp.61-70).

The identified and isolated gene can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Examples of vectors include, but arenot limited to, E. coli, bacteriophages such as lambda derivatives, orplasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g.,pGEX vectors, pmal-c, pFLAG, etc. The insertion into a cloning vectorcan, for example, be accomplished by ligating the DNA fragment into acloning vector which has complementary cohesive termini. However, if thecomplementary restriction sites used to fragment the DNA are not presentin the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. Recombinant molecules can be introduced into host cells viatransformation, transfection, infection, electroporation, etc., so thatmany copies of the gene sequence are generated. Preferably, the clonedgene is contained on a shuttle vector plasmid, which provides forexpansion in a cloning cell, e.g., E. coli, and facile purification forsubsequent insertion into an appropriate expression cell line, if suchis desired. For example, a shuttle vector, which is a vector that canreplicate in more than one type of organism, can be prepared forreplication in both E. coli and Saccharomyces cerevisiae by linkingsequences from an E. coli plasmid with sequences from the yeast 2μplasmid.

In an alternative method, the desired gene may be identified andisolated after insertion into a suitable cloning vector in a “shot gun”approach. Enrichment for the desired gene, for example, by sizefractionation, can be done before insertion into the cloning vector.

Expression of Transmembrane Activator and CAML Interactor Polypeptides

The nucleotide sequence coding for the TACI protein, or antigenicfragment, derivative or analog thereof, or a functionally activederivative, including a chimeric protein, thereof, can be inserted intoan appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. Such elements are termed herein a “promoter.”Thus, the nucleic acid encoding the TACI protein of the presentinvention is operationally associated with a promoter in an expressionvector of the invention. Both cDNA and genomic sequences can be clonedand expressed under control of such regulatory sequences. An expressionvector also preferably includes a replication origin.

The necessary transcriptional and translational signals can be providedon a recombinant expression vector, or they may be supplied by thenative gene encoding a TACI protein and/or its flanking regions.

As pointed out above, potential chimeric partners for TACI proteininclude those having lectin domains, either from naturally occurringmultivalent lectin receptors, such as mannose receptor of macrophages,natural lectins, or other sources, or a substitute cytoplasmic domain,capable of mediating signal transduction or modifying the endocyticprocessing.

Potential host-vector systems include but are not limited to mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

A recombinant TACI protein of the invention, or functional fragment,derivative, chimeric construct, or analog thereof, may be expressedchromosomally, after integration of the coding sequence byrecombination. In this regard, any of a number of amplification systemsmay be used to achieve high levels of stable gene expression (seeSambrook et al., 1989, supra).

The cell containing the recombinant vector comprising the nucleic acidencoding a TACI protein is cultured in an appropriate cell culturemedium under conditions that provide for expression of TACI protein bythe cell.

Any of the methods previously described for the insertion of DNAfragments into a cloning vector may be used to construct expressionvectors containing a gene consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombination (genetic recombination).

Expression of TACI protein may be controlled by any promoter/enhancerelement known in the art, but these regulatory elements must befunctional in the host selected for expression. Promoters which may beused to control TACI protein gene expression include, but are notlimited to, the SV40 early promoter region [Benoist and Chambon (1981)Nature 290:304-310], the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus [Yamamoto, et al. (1980) Cell 22:787-797],the herpes thymidine kinase promoter [Wagner et al. (1981) Proc. Natl.Acad. Sci. U.S.A., 78:1441-1445], the regulatory sequences of themetallothionein gene [Brinster et al. (1982) Nature 296:39-42];prokaryotic expression vectors such as the β-lactamase promoter[Villa-Kamaroff, et al. (1978) Proc. Natl. Acad. Sci. U.S.A.,75:3727-3731], or the tac promoter [DeBoer, et al. (1983) Proc. Natl.Acad. Sci. U.S.A. 80:21-25]; see also “Useful proteins from recombinantbacteria” in Scientific American (1980) 242:74-94; promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter; and the animal transcriptional control regions,which exhibit tissue specificity and have been utilized in transgenicanimals: elastase I gene control region which is active in pancreaticacinar cells [Swift et al. (1984) Cell 38:639-646]; [Ornitz et al.(1986) Cold Spring Harbor Symp. Quant. Biol. 50:399-409]; [MacDonald(1987) Hepatology 7:425-515]; insulin gene control region which isactive in pancreatic beta cells [Hanahan (1985) Nature 315:115-122],immunoglobulin gene control region which is active in lymphoid cells[Grosschedl et al. (1984) Cell 38:647-658]; [Adames et al. (1985) Nature318:533-538]; [Alexander et al. (1987) Mol. Cell. Biol. 7:1436-1444],mouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells [Leder et al. (1986) Cell 45:485-495],albumin gene control region which is active in liver [Pinkert et al.(1987) Genes and Devel. 1:268-276], alpha-fetoprotein gene controlregion which is active in liver [Krumlauf et al. (1985) Mol. Cell. Biol.5:1639-1648]; [Hammer et al. (1987) Science 235:53-58], alpha1-antitrypsin gene control region which is active in the liver [Kelseyet al. (1987) Genes and Devel. 1:161-171], beta-globin gene controlregion which is active in myeloid cells [Mogram et al. (1985) Nature315:338-340]; [Kollias et al. (1986) Cell 46:89-94], myelin basicprotein gene control region which is active in oligodendrocyte cells inthe brain [Readhead et al. (1987) Cell 48:703-712], myosin light chain-2gene control region which is active in skeletal muscle [Sani (1985)Nature 314:283-286], and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus [Mason et al. (1986) Science234:1372-1378].

Expression vectors containing a nucleic acid encoding a TACI protein ofthe invention can be identified by four general approaches: (a) PCRamplification of the desired plasmid DNA or specific mRNA, (b) nucleicacid hybridization, (c) presence or absence of selection marker genefunctions, and (d) expression of inserted sequences. In the firstapproach, the nucleic acids can be amplified by PCR to provide fordetection of the amplified product. In the second approach, the presenceof a foreign gene inserted in an expression vector can be detected bynucleic acid hybridization using probes comprising sequences that arehomologous to an inserted marker gene. In the third approach, therecombinant vector/host system can be identified and selected based uponthe presence or absence of certain “selection marker” gene functions(e.g., β-galactosidase activity, thymidine kinase activity, resistanceto antibiotics, transformation phenotype, occlusion body formation inbaculovirus, etc.) caused by the insertion of foreign genes in thevector. In another example, if the nucleic acid encoding a TACI proteinis inserted within the “selection marker” gene sequence of the vector,recombinants containing the TACI protein insert can be identified by theabsence of the TACI protein gene function. In the fourth approach,recombinant expression vectors can be identified by assaying for theactivity, biochemical, or immunological characteristics of the geneproduct expressed by the recombinant, provided that the expressedprotein assumes a functionally active conformation.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol El, pCR1, pBR322, pMal-C2, pET, pGEX [Smith et al. (1988) Gene67:31-40], pMB9 and their derivatives, plasmids such as RP4; phage DNAS,e.g., the numerous derivatives of phage λ, e.g., NM989, and other phageDNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmidssuch as the 2μ plasmid or derivatives thereof; vectors useful ineukaryotic cells, such as vectors useful in insect or mammalian cells;vectors derived from combinations of plasmids and phage DNAs, such asplasmids that have been modified to employ phage DNA or other expressioncontrol sequences; and the like.

For example, in a baculovirus expression systems, both non-fusiontransfer vectors, such as but not limited to pVL941 (BamH1 cloning site;Summers), pVL1393 (BamH1, SmaI, XbaI, EcoR1, NotI, XmaIII, BglII, andPstI cloning site; Invitrogen), pVL1392 (BglII, PstI, NotI, XmaIII,EcoRI, XbaI, SmaI, and BamH1 cloning site; Summers and Invitrogen), andpBlueBacIII (BamH1, BglII, PstI, NcoI, and HindIII cloning site, withblue/white recombinant screening possible; Invitrogen), and fusiontransfer vectors, such as but not limited to pAc700 (BamH1 and KpnIcloning site, in which the BamH1 recognition site begins with theinitiation codon; Summers), pAc701 and pAc702 (same as pAc700, withdifferent reading frames), pAc360 (BamH1 cloning site 36 base pairsdownstream of a polyhedrin initiation codon; Invitrogen (195)), andpBlueBacHisA, B, C (three different reading frames, with BamH1, BglII,PstI, NcoI, and HindIII cloning site, an N-terminal peptide for ProBondpurification, and blue/white recombinant screening of plaques;Invitrogen (220)) can be used.

Mammalian expression vectors contemplated for use in the inventioninclude vectors with inducible promoters, such as the dihydrofolatereductase (DHFR) promoter, e.g., any expression vector with a DHFRexpression vector, or a DHFR/methotrexate co-amplification vector, suchas pED (PstI, SalI, SbaI, SmaI, and EcoRI cloning site, with the vectorexpressing both the cloned gene and DHFR; see Kaufman (1991) CurrentProtocols in Molecular Biology 16:12. Alternatively, a glutaminesynthetase/methionine sulfoximine co-amplification vector, such as pEE14(HindIII, XbaI, SmaI, SbaI, EcoRI, and BclI cloning site, in which thevector expresses glutamine synthase and the cloned gene; Celltech). Inanother embodiment, a vector that directs episomal expression undercontrol of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamH1,SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site,constitutive RSV-LTR promoter, hygromycin selectable marker;Invitrogen), pCEP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII,and KpnI cloning site, constitutive hCMV immediate early gene,hygromycin selectable marker; Invitrogen), pMEP4 (KpnI, PvuI, NheI,HindIII, NotI, XhoI, SfiI, BamH1 cloning site, inducible metallothioneinIIa gene promoter, hygromycin selectable marker: Invitrogen), pREP8(BamH1, XhoI, NotI, HindIII, NheI, and KpnI cloning site, RSV-LTRpromoter, histidinol selectable marker; Invitrogen), pREP9 (KpnI, NheI,HindIII, NotI, XhoI, SfiI, and BamH1 cloning site, RSV-LTR promoter,G418 selectable marker; Invitrogen), and pEBVHis (RSV-LTR promoter,hygromycin selectable marker, N-terminal peptide purifiable via ProBondresin and cleaved by enterokinase; Invitrogen). Selectable mammalianexpression vectors for use in the invention include pRc/CMV (HindIII,BstXI, NotI, SbaI, and ApaI cloning site, G418 selection; Invitrogen),pRc/RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning site, G418 selection;Invitrogen), and others. Vaccinia virus mammalian expression vectors(see, Kaufman, 1991, supra) for use according to the invention includebut are not limited to pSC11 (SmaI cloning site, TO- and β-galselection), pMJ601 (SalI, SmaI, AflI, NarI, BspMII, BamHI, ApaI, NheI,SacI, KpnI, and HindIII cloning site; TK- and β-gal selection), andpTKgptF1S (EcoRI, PstI, SalI, AccI, HindIII, SbaI, BamHI, and Hpacloning site, TK or XPRT selection).

Yeast expression systems can also be used according to the invention toexpress the TACI protein. For example, the non-fusion pYES2 vector(XbaI, SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamH1, SacI, Kpn1, andHindIII cloning sit; Invitrogen) or the fusion pYESHisA, B, C (XbaI,SphI, ShoI, NotI, BstXI, EcoRI, BamH1, SacI, KpnI, and HindIII cloningsite, N-terminal peptide purified with ProBond resin and cleaved withenterokinase; Invitrogen), to mention just two, can be employedaccording to the invention.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Aspreviously explained, the expression vectors which can be used include,but are not limited to, the following vectors or their derivatives:human or animal viruses such as vaccinia virus or adenovirus; insectviruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g.,lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification (e.g., glycosylation,cleavage e.g., of signal sequence) of proteins. Appropriate cell linesor host systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed. For example, expression ina bacterial system can be used to produce an non-glycosylated coreprotein product.

Vectors are introduced into the desired host cells by methods known inthe art, e.g., transfection, electroporation, micro injection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al. (1992) J. Biol. Chem.267:963-967; Wu and Wu (1988) J. Biol. Chem. 263:14621-14624; Hartmut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

Gene Therapy and Transgenic Vectors

A genetic deficiency of a TACI protein can be one of the many factorsinvolved in inherited immunodeficiency. The present invention includesgene therapy with the TACI protein cDNA that restores normal lymphocytefunction in patients having a genetic defect in the TransmembraneActivator and CAML Interactor protein gene. The present invention alsoincludes modified forms of TACI to be used as gene-therapeutic toolsthrough inserting them into blood stem cells in preparation forre-infusion into patients as part of bone marrow transplant regimes. Inone embodiment an epitope tagged-TACI derivative is used as a way toselectively activate only those lymphocytes derived from the marrowtransplant by injection of the specific antibody into the patient. In analternative embodiment transduction of a TACI gene lacking itsintracellular portion is used to create a relatively innocuouslymphocyte with diminished responsiveness. This embodiment is useful inautoimmune diseases involving activation of B cells.

In one embodiment, a gene encoding a TACI protein or polypeptide domainfragment thereof is introduced in vivo in a viral vector. Such vectorsinclude an attenuated or defective DNA virus, such as but not limited toherpes simplex virus (HSV), papilloma virus, Epstein Barr virus (EBV),adenovirus, adeno-associated virus (AAV), and the like. Defectiveviruses, which entirely or almost entirely lack viral genes, arepreferred. Defective virus is not infective after introduction into acell. Use of defective viral vectors allows for administration to cellsin a specific, localized area, without concern that the vector caninfect other cells. Thus, lymphocytes can be specifically targeted.Examples of particular vectors include, but are not limited to, adefective herpes virus 1 (HSV1) vector [Kaplitt et al. (1991) Molec.Cell. Neurosci. 2:320-330], an attenuated adenovirus vector, such as thevector described by Stratford-PerricaudetI [(1992) J. Clin. Invest.90:626-630], and a defective adeno-associated virus vector [Samulski etal. (1987) J. Virol. 61:3096-3101]; [Samulski et al. (1989) J. Virol,63:3822-3828].

Preferably, for in vivo administration, an appropriate immunosuppressivetreatment is employed in conjunction with the viral vector, e.g.,adenovirus vector, to avoid immuno-deactivation of the viral vector andtransfected cells. For example, immunosuppressive cytokines, such asinterleukin-12 (IL-12), interferon-γ (IFN-γ), or anti-CD4 antibody, canbe administered to block humoral or cellular immune responses to theviral vectors [see, e.g., Wilson (1995) Nature Medicine]. In addition,it is advantageous to employ a viral vector that is engineered toexpress a minimal number of antigens.

In another embodiment the gene can be introduced in a retroviral vector,e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346; Mann etal. (1983) Cell 33:153; Temin et al., U.S. Pat. No. 4,650,764; Temin etal., U.S. Pat. No. 4,980,289; Markowitz et al. (1988) J. Virol. 62:1120;Temin et al., U.S. Pat. No. 5,124,263; International Patent PublicationNo. WO 95/07358, published Mar. 16, 1995, by Dougherty et al.; and Kuoet al. (1993) Blood 82:845.

Targeted gene delivery is described in International Patent PublicationWO 95/28494, published October 1995.

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker [Felgneret. al. (1987) Proc. Natl. Acad. Sci. USA. 84:7413-7417; see Mackey, etal. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031]. The use ofcationic lipids may promote encapsulation of negatively charged nucleicacids, and also promote fusion with negatively charged cell membranes[Felgner and Ringold (1989) Science 337:387-388]. The use of lipofectionto introduce exogenous genes into the specific organs in vivo hascertain practical advantages. Molecular targeting of liposomes tospecific cells represents one area of benefit. It is clear thatdirecting transfection to particular cell types would be particularlyadvantageous in a tissue with cellular heterogeneity, such as pancreas,liver, kidney, and the brain. Lipids may be chemically coupled to othermolecules for the purpose of targeting [see Mackey, et al., supra].Targeted peptides, e.g., hormones or neurotransmitters, and proteinssuch as antibodies, or non-peptide molecules could be coupled toliposomes chemically.

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced into thedesired host cells by methods known in the art, e.g., transfection,electroporation, micro injection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter [see, e.g., Wu et al. (1992) J. Biol. Chem.267:963-967; Wu and Wu (1988) J. Biol. Chem., 263:14621-14624; Hartmutet al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990].

In a preferred embodiment of the present invention, a gene therapyvector as described above employs a transcription control sequenceoperably associated with the sequence for the TACI protein inserted inthe vector. That is, a specific expression vector of the presentinvention can be used in gene therapy.

Gene Targeting

As used herein “Gene targeting” is a type of homologous recombinationthat occurs when a fragment of genomic DNA is introduced into amammalian cell and that fragment locates and recombines with endogenoushomologous sequences.

As used herein a “knockout mouse” is a mouse that contains within itsgenome a specific gene that has been inactivated by the method of genetargeting. A knockout mouse includes both the heterozygote mouse (i.e.,one defective allele and one wild-type allele) and the homozygous mutant(i.e., two defective alleles).

As used herein a “marker gene” is a selection marker that facilitatesthe isolation of rare transfected cells from the majority of treatedcells in the population. A non-comprehensive list of such markersincludes neomycin phosphotransferase, hygromycin B phosphotransferase,Xanthine/guanine phosphoribosyl transferase, herpes simplex thymidinekinase, and diphtheria toxin.

The functional activity of Transmembrane Activator and CAML Interactorprotein can be evaluated transgenically. In this respect, a transgenicmouse model can be used. The Transmembrane Activator and CAML Interactorprotein gene can be used in complementation studies employing atransgenic mouse. Transgenic vectors, including viral vectors, or cosmidclones (or phage clones) corresponding to the wild type locus of acandidate gene, can be constructed using the isolated TACI protein gene.Cosmids may be introduced into transgenic mice using publishedprocedures [Jaenisch (1988) Science 240:1468-1474]. In a genetic sense,the transgene acts as a suppressor mutation.

Alternatively, a transgenic animal model can be prepared in whichexpression of the TACI protein gene is disrupted. Gene expression isdisrupted, according to the invention, when no functional protein isexpressed. One standard method to evaluate the phenotypic effect of agene product is to employ knock-out technology to delete the gene.Alternatively, recombinant techniques can be used to introducemutations, such as nonsense and amber mutations, or mutations that leadto expression of an inactive protein. In another embodiment, TACIprotein genes can be tested by examining their phenotypic effects whenexpressed in antisense orientation in wild-type animals. In thisapproach, expression of the wild-type allele is suppressed, which leadsto a mutant phenotype. RNA×RNA duplex formation (antisense-sense)prevents normal handling of mRNA, resulting in partial or completeelimination of wild-type gene effect. This technique has been used toinhibit TK synthesis in tissue culture and to produce phenotypes of theKruppel mutation in Drosophila, and the Shiverer mutation in mice [Izantet al. Cell (1984) 36:1007-1015; Green et al. (1986) Annu. Rev. Biochem.55:569-597; Katsuki et al. (1988) Science 241:593-595]. An importantadvantage of this approach is that only a small portion of the gene needbe expressed for effective inhibition of expression of the entirecognate mRNA. The antisense transgene will be placed under control ofits own promoter or another promoter expressed in the correct cell type,and placed upstream of the SV40 polyA site. This transgene will be usedto make transgenic mice, or by using gene knockout technology.

Thus the present invention extends to the preparation of antisensenucleotides and ribozymes that may be used to interfere with theexpression of the TACI protein at the translational level. This approachutilizes antisense nucleic acid and ribozymes to block translation of aspecific mRNA, either by masking that mRNA with an antisense nucleicacid or cleaving it with a ribozyme. Genes encoding TACI mRNA-specificantisense or ribozyme nucleic acids can be introduced, e.g., usingtechniques as described above for “Gene Therapy.” Alternatively,synthetic antisense or ribozyme oligonucleotides can be prepared.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule [see Marcus-Sekura(1988) Anal. Biochem. 172:298]. In the cell, they hybridize to thatmRNA, forming a double stranded molecule. The cell does not translate anmRNA in this double-stranded form. Therefore, antisense nucleic acidsinterfere with the expression of mRNA into protein. Oligomers of aboutfifteen nucleotides and molecules that hybridize to the AUG initiationcodon will be particularly efficient, since they are easy to synthesizeand are likely to pose fewer problems than larger molecules whenintroducing them into organ cells. Antisense methods have been used toinhibit the expression of many genes in vitro [Marcus-Sekura (1988),supra; Hambor et al. (1988) J. Exp. Med. 168:1237]. Preferably syntheticantisense nucleotides contain phosphoester analogs, such asphosphorothiolates, or thioesters, rather than natural phophoesterbonds. Such phosphoester bond analogs are more resistant to degradation,increasing the stability, and therefore the efficacy, of the antisensenucleic acids.

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Investigators haveidentified two types of ribozymes, Tetrahymena-type and“hammerhead”-type. Hammerhead-type ribozymes are preferable toTetrahymena-type ribozymes for inactivating a specific mRNA species, andeighteen base recognition sequences are preferable to shorterrecognition sequences.

The DNA sequences encoding the TACI protein described and enabled hereinmay thus be used to prepare antisense molecules against and ribozymesthat cleave mRNAs for the TACI protein, thus inhibiting expression ofthe gene encoding the TACI protein, which may reduce the level of immunestimulation by dendritic cells, or the level of clonal detection bymediated by thymic epithelial cells.

Gene targeting in embryonic stem cells is a relatively new techniquethat allows the precise manipulation of genes in vivo. This techniqueallows the creation of mice with defined mutations in the structure ofany given gene. This ability to generate predetermined mutations givesinvestigators the ability to apply the power of genetics to the complexhuman immune system, as it has successfully been applied in neuronalsystems for such organisms as D. melanogaster and C. elegans.

A key to finding treatments for many disorders has been the developmentof appropriate animal models. The present invention includes a knockoutmouse containing a non-functional allele for the gene that naturallyencodes and expresses functional TACI protein. Included within thisaspect of the invention is a knockout mouse containing twonon-functional alleles for the gene that naturally encodes and expressesfunctional TACI protein, and therefore is unable to express functionalTACI protein.

Non-functional alleles can be generated in any number of ways that arewell known in the art, all of which may be used in the presentinvention. In some embodiments, a non-functional allele is madedefective by an insertion of extraneous DNA into the coding region ofTACI protein allele. In a preferred embodiment, the insertion is placedin the first exon of the coding region of the TACI protein gene. In morepreferred embodiments, the insertion contains a signal to terminatetranscription prior to the transcription of a region of the allele thatencodes TACI protein. In these preferred embodiments it is still morepreferred to remove a section of DNA at the beginning of the codingregion for the TACI protein and replacing it with the above insertion.

The present invention also includes a method for producing the knockoutmouse of the instant invention that includes: obtaining genomic DNAencoding a TACI protein, constructing a vector containing said genomicDNA and a marker gene wherein said marker gene is placed within the exonof said genomic DNA. The vector is then electroporated into an embryonicstem cell and an embryonic stem cell is selected that has integrated thevector into the genome, wherein the selected cell has integrated themarker gene into the endogenous site of the gene for the TACI protein inthe mouse genome. The cell is then injected into a mouse blastocystwhich is then re-implanted into a pseudopregnant female mouse, whichgives birth to a chimeric mouse containing a defective allele for theTACI protein in its germ line. The chimeric mouse is then mated to amouse of a standard in-bred line to generate a heterozygous knockoutmouse. Two heterozygous mice are then bred generating a homozygousknockout mouse offspring. Detailed protocols for successful genetargeting are well known in the art, and for example, as described byJoyner, A. L. (1993) Gene Targeting: A Practical Approach. The PracticalApproach Series (Rickwood, D. and Hames, B. D., Eds.), IRL Press, Oxfordwhich is hereby incorporated by reference in its entirety.

Another aspect of the invention is a method for selecting a therapeuticagent for possible use as an immunosuppressant which comprisesadministering a suspected therapeutic agent to the knockout mouse of thepresent invention and measuring and/or determining the putativetherapeutic agent's effect on any of the phenotypic characteristicswhich may be believed to be related to the immunodeficiency.

A preferred embodiment of this aspect of the invention includesadministering a suspected therapeutic agent to the knockout mouse of thepresent invention and measuring a test response in the knockout mouse,wherein the normal response of the knockout mouse in the absence of atherapeutic agent is characteristically different from that of wild-typemice. The potential therapeutic agents are selected on the basis ofwhether there is a statistical significance between test response andthe normal response. Potential therapeutic agents are selected that showa statistically significant change in the characteristicmeasured/determined. In a preferred embodiment, the normal response ofthe knockout mouse in the absence of a therapeutic agent ischaracteristically different by being characteristically lower than thatof wild-type mice and the selected therapeutic agents act to raise thesensitivity of that characteristic.

The suspected therapeutical agents may be obtained from any number ofdrug or peptide libraries including those commercially available fromdrug chemical companies.

Purification of TACI Proteins and Homologues Thereof:

The TACI protein of the present invention and homologues thereof can bepurified by any number of procedures that encompass a wide variety ofknown purification steps. Those with skill in the art would know torefer to references, such as the Methods of Enzymology series, forgreater detail and breadth. Initial steps for purifying the proteins ofthe present invention include salting in or salting out, such as inammonium sulfate fractionations; solvent exclusion fractionations, e.g.,an ethanol precipitation; detergent extractions to free membrane boundproteins using such detergents as Triton X-100, Tween-20 etc.; or highsalt extractions. Solubilization of proteins may also be achieved usingaprotic solvents such as dimethyl sulfoxide and hexamethylphosphoramide.In addition, high speed ultracentrifugation may be used either alone orin conjunction with other extraction techniques.

Generally good secondary isolation or purification steps include solidphase absorption using calcium phosphate gel or hydroxyapatite; or solidphase binding, Solid phase binding may be performed through ionicbonding, with either an anion exchanger, such as diethylaminoethyl(DEAE), or diethyl[2-hydroxy propyl]amino ethyl (QAE) Sephadex orcellulose; or with a cation exchanger such as carboxymethyl (CM) orsulfo propyl (SP) Sephadex or cellulose. Alternative means of solidphase binding includes the exploitation of hydrophobic interactionse.g., the using of a solid support such as phenylSepharose and a highsalt buffer; affinity-binding, using, e.g., CAML (or a TACI-bindingfragment thereof) bound to an activated support; immuno-binding, usinge.g., an antibody to the TACI protein bound to an activated support; aswell as other solid phase supports including those that contain specificdyes or lectins etc. A further solid phase support technique that isoften used at the end of the purification procedure relies on sizeexclusion, such as Sephadex and Sepharose gels, or pressurized orcentrifugal membrane techniques, using size exclusion membrane filters.

Solid phase support separations are generally performed batch-wise withlow-speed centrifugations or by column chromatography. High performanceliquid chromatography (HPLC), including such related techniques as FPLC,is presently the most common means of performing liquid chromatography.Size exclusion techniques may also be accomplished with the aid of lowspeed centrifugation.

In addition size permeation techniques such as gel electrophoretictechniques may be employed. These techniques are generally performed intubes, slabs or by capillary electrophoresis.

Almost all steps involving protein purification employ a biologicalbuffer at a pH close to the pKa of that buffer. Typical buffers can bepurchased from most biochemical catalogues and include the classicalbuffers such as Tris, pyrophosphate, monophosphate, and diphosphate, orthe Good buffers [Good et al. (1966) Biochemistry 5:467]; [Good, N. E.and Izawa, S. (1972) Meth. Enzymol. 24, Part B, 53]; and [Fergunson, W.J. and Good, N. E. (1980) Anal. Biochem. 104:300] such as Mes, Hepes,Mops, tricine and Ches.

Materials to perform all of these techniques are available from avariety of sources such as Sigma Chemical Company in St. Louis, Mo.

Antibodies to the TACI Protein

The present invention discloses the protein sequence and properties of aspecific Transmembrane Activator and CAML Interactor protein, TACI,thereby enabling the development of antibody reagents specific for theextracellular portion of the receptor. Polyvalent antibody reagents canact to cross-link and activate TACI signaling in lymphocytes, a usefulprocess in situations where enhanced lymphocyte responsiveness isbeneficial. Such situations include for example, in the treatment ofimmunodeficiencies that are either congenital or acquired e.g., AIDS. Inaddition, these antibody reagents may be used as an adjuvant treatmentin cancer in instances that the immune system can aid in the eradicationof neoplastic cells. Similarly monovalent antibody reagents can act toblock access to TACI in lymphocytes, a process that is useful insituations where depressed lymphocyte responsiveness is beneficial suchas during organ transplants.

According to the present invention, the TACI protein produced naturally,recombinantly or by chemical synthesis, and fragments or otherderivatives or analogs thereof, including fusion proteins, may be usedas an immunogen to generate antibodies that recognize the TACI protein.Such antibodies include but are not limited to polyclonal, monoclonal,chimeric, single chain, Fab fragments, and an Fab expression library.The anti-TACI protein antibodies of the invention may be cross reactive,e.g., they may recognize the TACI protein from different species.Polyclonal antibodies have greater likelihood of cross reactivity.Alternatively, an antibody of the invention may be specific for a singleform of the TACI protein. Preferably, such an antibody is specific forhuman TACI protein. Antibodies of the invention can be labeled, asdescribed above for TACI proteins and polypeptides.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to the TACI protein or derivative or analogthereof. For the production of antibody, various host animals can beimmunized by injection with the TACI protein, or a derivative (e.g.,fragment or fusion protein) thereof, including but not limited torabbits, mice, rats, sheep, goats, etc. In one embodiment, the TACIprotein or fragment thereof can be conjugated to an immunogenic carrier,e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the TACIprotein, or fragment, analog, or derivative thereof, any technique thatprovides for the production of antibody molecules by continuous celllines in culture may be used. These include but are not limited to thehybridoma technique originally developed by Kohler and Milstein [(1975)Nature 256:495-497], as well as the trioma technique, the human B-cellhybridoma technique [Kozbor et al. (1983) Immunology Today 4:72]; [Coteet al. (1983) Proc. Natl. Acad. Sci. USA. 80:2026-2030], and theEBV-hybridoma technique to produce human monoclonal antibodies [Cole etal. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96]. In an additional embodiment of the invention, monoclonalantibodies can be produced in germ-free animals utilizing recenttechnology [PCT/US90/02545]. In fact, according to the invention,techniques developed for the production of “chimeric antibodies”[Morrison et al. (1984) J. Bacteriol. 159:870; Neuberger et al. (1984)Nature 312:604-608; Takeda et al. (1985) Nature 314:452-454] by splicingthe genes from a mouse antibody molecule specific for a TACI proteintogether with genes from a human antibody molecule of appropriatebiological activity can be used; such antibodies are within the scope ofthis invention. Such human or humanized chimeric antibodies arepreferred for use in therapy of human diseases or disorders (describedinfra), since the human or humanized antibodies are much less likelythan xenogeneic antibodies to induce an immune response, in particularan allergic response, themselves.

According to the invention, techniques described for the production ofsingle chain antibodies [U.S. Pat. Nos. 5,476,786 and 5,132,405 toHuston; U.S. Pat. No. 4,946,778] can be adapted to produce the TACIprotein-specific single chain antibodies. An additional embodiment ofthe invention utilizes the techniques described for the construction ofFab expression libraries [Huse et al. (1989) Science 246:1275-1281] toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity for a TACI protein, or its derivatives, or analogs.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of a TACI protein, one may assay generated hybridomasfor a product which binds to a TACI protein fragment containing suchepitope. For selection of an antibody specific to a TACI protein from aparticular species of animal, one can select on the basis of positivebinding with the TACI protein expressed by or isolated from cells ofthat species of animal.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the TACI protein, e.g., forWestern blotting, imaging the TACI protein in situ, measuring levelsthereof in appropriate physiological samples, etc. using any of thedetection techniques mentioned above or known in the art.

In a specific embodiment, antibodies that agonize or antagonize theactivity of the TACI protein can be generated. Such antibodies can betested using the assays described infra for identifying ligands.

Ligands to the TACI Protein

The identity of the endogenous ligand of the TACI protein is unknown.The TACI protein can be used, to screen clones in order to identify theendogenous ligand(s). This ligand is likely to be involved in theregulation of the immune system as well, and thus should have similar orcomplementary uses to those described herein. Methods for screening forTACI-1 ligands include: (i) through the use of a yeast two-hybrid systemwith TACI-1 as “bait”, as described, e.g., in Chien et al. (1991) Proc.Natl. Science, USA, 88:9578-9582 and Durfee et al. (1993) Genes Dev.7:555-69; (ii) interaction cloning from E. coli expression-libraries asdescribed above; and (iii) functional expression cloning in mammaliancells as described above.

Identification and isolation of a gene encoding a TACI protein of theinvention provides for expression of the TACI protein or fragmentsthereof in quantities greater than can be isolated from natural sources,or in cells that are specially engineered to be regulated by the TACIprotein expressed after transfection or transformation of the cells.Accordingly, in addition to rational design of agonists and antagonistsbased on the structure of the TACI protein, the present inventioncontemplates an alternative method for identifying specific ligands ofthe TACI protein using various screening assays known in the art.

Any screening technique known in the art can be used to screen for TACIprotein agonists or antagonists. The present invention contemplatesscreens for small molecule ligands or ligand analogs and mimics, as wellas screens for the natural ligand(s) that bind to and agonize orantagonize the TACI protein in vivo. For example, natural productslibraries can be screened using assays of the invention for moleculesthat agonize or antagonize the TACI protein activity, or that bind tothe extracellular domain or cytoplasmic domain of TACI.

Knowledge of the primary sequence of the TACI protein, and thesimilarity of that sequence with proteins of known function, can providean initial clue as the inhibitors or antagonists of the protein.Identification and screening of antagonists is further facilitated bydetermining structural features of the protein, e.g., using X-raycrystallography, neutron diffraction, nuclear magnetic resonancespectrometry, and other techniques for structure determination. Thesetechniques provide for the rational design or identification of agonistsand antagonists.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” [Scott and Smith (1990) Science249:386-390; Cwirla, et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Devlin et al. (1990) Science 249:404-406], very large libraries can beconstructed (10⁶-10⁸ chemical entities). A second approach usesprimarily chemical methods, of which the Geysen method [Geysen et al.(1986) Molecular Immunology 23:709-715; Geysen et al. (1987) J.Immunologic Method 102:259-274] and the method of Fodor et al. [(1991)Science 251:767-773] are examples. Furka et al. [14th InternationalCongress of Biochemistry, Volume 5, Abstract FR:013 (1988); Furka (1991)Int. J. Peptide Protein Res. 37:487-493], Houghton [U.S. Pat. No.4,631,211, issued December 1986] and Rutter et al. [U.S. Pat. No.5,010,175, issued Apr. 23, 1991] describe methods to produce a mixtureof peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries [Needels et al. (1993) Proc.Natl. Acad. Sci. USA 90:10700-4; Ohlmeyer et al. (1993) Proc. Natl.Acad. Sci. USA 90:10922-10926; Lam et al., International PatentPublication No. WO 92/00252; Kocis et al., International PatentPublication No. WO 9428028, each of which is incorporated herein byreference in its entirety], and the like can be used to screen for theTACI protein ligands according to the present invention.

Alternatively, assays for binding of soluble ligand to cells thatexpress recombinant forms of the TACI N-Terminal extracellular domaincan be performed. The soluble ligands can be provided readily asrecombinant or synthetic polypeptides.

The screening can be performed with recombinant cells that express TACI,or a fragment thereof, or alternatively, using purified protein, e.g.,produced recombinantly, as described above. For example, the ability oflabeled, soluble or solubilized TACI fragment to bind ligand can be usedto screen libraries, as described in the foregoing references.

Screening For Novel Immunosuppressant Drugs

Certain diseases result from over activation of the B-lymphocyteresponse. One example is Systemic Lupus Erythematosus (SLE), in whichantibodies are created that react with antigens (proteins, DNA, etc.)naturally present in the patient. The antibodies form complexes with theantigens, and circulate as reactive protein agglomerates. Thesecomplexes deposit in different organs and lead to the many symptoms ofSLE, including kidney failure, neurologic symptoms, and death. Currenttreatments include the use of relatively non-specific immunosuppressantssuch as cyclosporin A or steroids which suppress responses in both T andB cells. Although they can often effectively treat SLE and similardiseases, there is a significant risk of over-immunosuppression, inwhich the patient develops serious infections because of lack offunctioning T-cells. A new immunosuppressant drug that selectivelyblocks the action of B-lymphocytes, while leaving T-cells intact toprotect patients from viral pathogens would be extremely useful to treatthese diseases. Therefore, TACI-1 can be used as a novel tool fordeveloping immunosuppressant drugs specific for B-lymphocytes.

The TACI protein is not naturally present in mature T-lymphocytes orJurkat T cells, a cell line that has phenotypic characteristics ofmature T-cells. Indeed, the highest level of expression of the TACIprotein is on peripheral B lymphocytes, whereas peripheral T-cells donot express the TACI protein. However, Jurkat T cells can be transfectedwith a TACI expression plasmid and the TACI expressed can be readilystimulated by cross linking to a TACI specific antibody. Thisstimulation leads to the activation of a pair of second messengerpathways that are both necessary and sufficient to stimulate the earlyT-cell transcription factor NF-AT.

In a particular embodiment Jurkat T cells are transfected with TACI-1expression plasmid and a NF-AT-reporter plasmid. Jurkat T cellsnaturally express the T-cell receptor (TCR). The cells are stimulated bythe addition of antibodies to TACI-1, antibodies to TCR, or antibodiesto both. Candidate drugs are mixed with the Jurkat T cells and theeffect of these drugs is determined. Phage and chemical librariesdescribed above may be used as sources for drug candidates.

These candidate drugs are then applied in a parallel experiment in whichantibodies to TCR are used in place of the antibodies to TACI-1. If thecandidate drug has no effect on the SEAP signal stimulation due to theantibody-dependent activation of TCR, the candidate drug is identifiedas having selective inhibition of the TACI-1 activated response. Suchselected drugs include that class of drugs which can selectively inhibitthe B-cell (antibody producing) response, while allowing T-cell mediated(cellular) immunity to proceed. Such selected drugs may be used to treatthe autoimmune diseases described above. Because TACI-1 activateslymphocytes by a novel mechanism (i.e. through direct contact with CAML)it is likely that a significant number of drugs are discoverable thatcan interfere with the CAML-dependent pathway, yet leave normalsignaling through the T-cell receptor intact.

In a preferred embodiment of this type, the NF-AT-reporter plasmidcontains the SEAP reporter. This signal is used to measure the degree ofinhibition of activation. The SEAP reporter assay can be scaled up so asto be performed by a robot screening apparatus. Drugs that block theactivation of TACI-1 but not TCR as measured by the SEAP reporter assayare identified as having selective inhibition of the TACI-1 activatedresponse.

Alternatively, a phage library can be employed. Phage libraries havebeen constructed which when infected into host E. coli produce randompeptide sequences of approximately 10 to 15 amino acids [Parmley andSmith (1988) Gene 73:305-318, Scott and Smith (1990) Science249:386-249]. Specifically, the phage library can be mixed in lowdilutions with permissive E. coli in low melting point LB agar which isthen poured on top of LB agar plates. After incubating the plates at 37°C. for a period of time, small clear plaques in a lawn of E. coli willform which represents active phage growth and lysis of the E. coli. Arepresentative of these phages can be absorbed to nylon filters byplacing dry filters onto the agar plates. The filters can be marked fororientation, removed, and placed in washing solutions to block anyremaining absorbent sites. The filters can then be placed in a solutioncontaining, for example, a radioactive fragment of a TACI proteincontaining the N-terminal extracellular domain e.g., for human TACI-1 itis a peptide having the amino acid sequence of SEQ ID NO:6. After aspecified incubation period, the filters can be thoroughly washed anddeveloped for autoradiography. Plaques containing the phage that bind tothe radioactive N-terminal extracellular domain can then be identified.These phages can be further cloned and then retested for their abilityto inhibit the stimulation of TACI by an anti-TACI antibody while notinhibiting the corresponding stimulation of TCR as described above.

Once the phages have been purified, the binding sequence containedwithin the phage can be determined by standard DNA sequencingtechniques. Once the DNA sequence is known, synthetic peptides can begenerated which represents these sequences.

The effective peptide(s) can be synthesized in large quantities for usein in vivo models and eventually in humans as immunosuppressant drugs,for example. It should be emphasized that synthetic peptide productionis relatively non-labor intensive, easily manufactured, qualitycontrolled and thus, large quantities of the desired product can beproduced quite cheaply. Similar combinations of mass produced syntheticpeptides have recently been used with great success [Patarroyo, (1990)Vaccine 10:175-178].

Therapeutic Methods and Compositions

The Transmembrane Activator CAML Interactor protein of the presentinvention can activate two signals normally used to initiate cell growthand division. This receptor is likely to be involved in the neoplastictransformation of T or B lymphocytes in lymphoma or leukemia. Therefore,dominant negative forms of TACI-1 are useful to suppress growth of suchcancer cells. Alternatively, TACI-1 over-stimulation can lead toprogrammed cell death. Taking advantage of this property, leukemia orlymphomas with TACI-1 cell surface expression may be induced to die bysuch over-stimulation. Activation of the TACI protein with antibody orcrosslinking may activate an endogenous pathway leading to apoptosis.Binding with a monomeric form of an antibody or analogous ligand canblock the TACI protein-associated endogenous pathway and interfere withgrowth simulation.

Therapeutic Stimulation of TACI Activity.

As discussed above, the present invention advantageously provides forselective stimulation of the immune system by agonizing TACI activity.TACI agonists include the TACI ligand or ligands discovered as describedsupra, and antibodies that crosslink the receptor. Ligand agonists orantibody agonists can be administered as described below for treatmentof subjects in whom immune stimulation, particularly of B cells, isdesired.

B cell responses can be particularly important in fighting infectiousdiseases, including, but not limited to, bacterial, viral, protozoan,and parasitic infections. Antibodies against infectious microorganismscan rapidly immobilize the organisms by binding to antigen, followed bycomplement attack or cell mediated attack. Thus, a TACI agonist of theinvention can be provided to a subject who is suffering from aninfection to boost humoral immune responses. TACI agonists may beparticularly useful for boosting immune responses after vaccination,during challenge with the infectious organism. Thus, subjectsparticularly at risk for infectious diseases, such as influenza, cansupplement a vaccination or memory immunity with a TACI agonist duringthe flu season. Comparable immune boosting could be used with subjectswho are entering or reentering an area with an endemic infectiousdisease, such as malaria.

In addition, B cell responses may be important in amplifying immuneresponses to tumors that express tumor-specific antigens. Thus, a TACIagonist may be provided where endogenous anti-tumor antibodies aredetected. Indeed, B cells or plasma cells expressing anti-tumor cellsurface immunoglobulin can be selected, such as by panning, andtransduced with TACI to allow for an amplification of their antibodyproduction.

In addition to amplifying beneficial immune responses, the present TACIagonists, e.g., ligands and antibodies, can be used to over-stimulatecells, such as B cells tumors (multiple myelomas, lymphomas, andleukemias), immature T cell tumors (leukemias and thymomas), andautoimmune or inflammatory cells and induce apoptosis in such cells,thereby reducing or eliminating the cancer or autoimmune/inflammatorycondition.

Therapies for Boosting Cellular Immune Responses.

Although TACI is found in only a subset of immature T cells, mature Tcells can be transfected or transduced in vivo or ex vivo to express afunctional TACI receptor, to amplify cellular immune responses.Preferably, tumor infiltrating cells are selected for such boosting, andreintroduced into the subject to more vigorously fight the tumor.

Therapeutic Methods by Antagonizing TACI Activity.

As discussed above, the present invention contemplates inhibiting TACIactivity by various means, including but not limited to use of the freeN-terminal extracellular domain; expression of a non-functionalextracellular domain lacking a signal transduction domain, e.g.,GPI-linked N-terminal TACI; use of antisense or ribozyme technologies tosuppress expression of TACI; and use of TACI antagonist ligands orantibodies.

Suppression of TACI activity is useful for treating undesirable immuneresponses, including autoimmune and inflammatory diseases,transplantation rejection, and graft-versus host (GVH) disease.Autoimmune and inflammatory diseases include immune complex-inducedvasculitis [Cochrane, (1984) Springer Seminar Immunopathol. 7:263],glomerulonephritis [Couser et al. (19815) Kidney Inst. 29:879],hemolytic anemia [Schreiber and Frank (1972) J. Clin. Invest. 51:575],myasthenia gravis [Lennon, et al. (1978) J. Exp. Med. 147:973; Bieseckerand Gomez (1989) J. Immunol. 142:2654], type II collagen-inducedarthritis [Watson and Townes (1985) J. Exp. Med. 162:1878]; experimentalallergic and hyperacute xenograft rejection [Knechtle et al. (1985) J.Heart Transplant 4(5):541; Guttman (1974) Transplantation 17:383; Adachiet al. (1987) Trans. Proc. 19(1):1145]; rheumatoid arthritis, andsystemic lupus erythematosus (SLE).

In another embodiment, where a lymphocyte cancer such as myeloma,lymphoma, or leukemia expresses TACI, and TACI contributes to cancergrowth, use of a TACI antagonist of the invention can be used tosuppress the cancer cell growth.

Accordingly, a component of a therapeutic composition such as apolyvalent or mono-valent antibody of the present invention may beintroduced parenterally, transmucosally, e.g., orally, nasally, orrectally, or transdermally. Preferably, administration is parenteral,e.g, via intravenous injection, and also including, but is not limitedto, intra-arteriole, intramuscular, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial administration.

In another embodiment, the therapeutic compound can be delivered in avesicle, in particular a liposome [see Langer (1990) Science249:1527-1533; Treat et al. (1989) Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss:New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.]. To reduce its systemic side effects, this may be apreferred method for introducing TACI.

In yet another embodiment, the therapeutic compound can be delivered ina controlled release system. For example, an agonist or antagonist tothe transmembrane binding protein of the present invention, such as anantibody, may be administered using intravenous infusion, an implantableosmotic pump, a transdermal patch, liposomes, or other modes ofadministration. In one embodiment, a pump may be used [see Langer,supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng 14:201; Buchwald et al.(1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574].In another embodiment, polymeric materials can be used [see (1974)Medical Applications of Controlled Release, Langer and Wise (eds.), CRCPress: Boca Raton, Fla.; (1984) Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball (eds.), Wiley: New York;Ranger and Peppas (1983) J. Macromol. Sci. Rev. Macromol. Chem. 23:61;see also Levy et al. (1985) Science 228:190; During et al. (1989) Ann.Neurol. 25:351; Howard et al. (1989) J. Neurosurg, 71:105]. In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose [see, e.g., Goodson (1984) MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138].Preferably, a controlled release device is introduced into a subject inproximity of the site of inappropriate immune activation or a tumor.

Other controlled release systems are discussed in the review by Langer(1990) Science 249:1527-1533.

In a further aspect, recombinant cells that have been transformed withthe TACI protein gene and that express high levels of the polypeptidecan be transplanted in a subject in need of the TACI protein. Preferablyautologous cells transformed with TACI protein are transplanted to avoidrejection.

The therapeutic compounds of the present invention can be delivered byintravenous, intraarterial, intraperitoneal, intramuscular, orsubcutaneous routes of administration. Alternatively, these compounds,properly formulated, can be administered by nasal or oraladministration. A constant supply of these therapeutic compounds can beensured by providing a therapeutically effective dose (i.e., a doseeffective to induce metabolic changes in a subject) at the necessaryintervals, e.g., daily, every 12 hours, etc. These parameters willdepend on the severity of the disease condition being treated, otheractions, such as diet modification, that are implemented, the weight,age, and sex of the subject, and other criteria, which can be readilydetermined according to standard good medical practice by those of skillin the art.

A subject in whom administration of these therapeutic agents is aneffective therapeutic regiment for an immunodeficiency disease ispreferably a human, but can be any animal. Thus, as can be readilyappreciated by one of ordinary skill in the art, the methods andpharmaceutical compositions of the present invention are particularlysuited to administration to any animal, particularly a mammal, andincluding, but by no means limited to, domestic animals, such as felineor canine subjects, farm animals, such as but not limited to bovine,equine, caprine, ovine, and porcine subjects, wild animals (whether inthe wild or in a zoological garden), research animals, such as mice,rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avian species, suchas chickens, turkeys, songbirds, etc., i.e., for veterinary medical use.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

Example Calcium Signaling by a Lymphocyte Surface Receptor MediatedThrough CAML Introduction

Ca²⁺ influx is a key regulator of antigen-stimulated lymphocyteactivation [Imboden et al. (1985) Immunol. 134:663-665]; [Crabtree &Clipstone (1994) Annu. Rev. Biochem. 63:1045-1083]; [Weiss & Littman(1994) Cell 76:263-274]. The CAML protein has been identified as aregulator of Ca²⁺ signaling that is necessary but not sufficient for theactivation of lymphocyte transcription factor, NF-AT (Bram & Crabtree,1994, supra). The location of CAML in cytoplasmic vesicles is consistentwith it regulating Ca²⁺ influx by modulating intracellular Ca²⁺ release.Here a novel human CAML-interacting receptor expressed by B lymphocytesthat acts as a cell-surface signaling molecule is disclosed. Thisreceptor, TACI (Iransmembrane Activator CAML Interactor), initiatesCa²⁺-dependent activation of NF-AT when cross linked with an antibody.The signal can be blocked by a dominant negative mutant of CAML. Asshown herein, the TACI protein also can independently activate the AP-1transcription factor, thus providing both signals required forlymphocyte activation. The TACI protein initiates a novel signaltransduction mechanism directly linking cell surface stimuli to theintracellular signaling molecule, CAML, and thereby defines a new classof lymphocyte-specific cell surface receptors that modulate the immuneresponse. In addition, the TACI protein is a novel tool that can be usedto regulate the immune system in either a positive or a negativedirection. TACI-1 is the human homologue of TACI illustrated in theinstant example.

Materials And Methods Molecular Cloning and Screening.

A human B-lymphocyte cDNA library is screened by the two-hybrid system[Fields & Song (1989) Nature 340:245-246]; [Durfee et al. (1993) GenesDev. 7:555-569], with the full coding region of CAML used as bait. Thehuman CAML cDNA is inserted into the yeast two-hybrid bait vector pAS1.This construct directs the expression of a GAL4-DNA binding domain fusedto the entire protein sequence of CAML. A B-lymphocyte library inplasmid pACT is transformed into yeast Y153 and potential interactingplasmids are identified by growth of colonies on media lacking histidineand containing 3-amino triazole.

One clone (TACI-1), out of eight primary positives was identified. TheTACI-1 cDNA is subcloned into a mammalian expression plasmid which addsan epitope tag to the amino-terminal end of the expressed protein. Thisconstruct is then transfected into the Jurkat T-lymphocyte cell line,COS cells, or NIH3T3 cells. In each case, cell surface expression of theTACI protein is demonstrated. The orientation of the protein is with theN-terminus outside the cell, as the epitope is available for reactionwith a specific antibody even without permeablizing the cell membrane.This result facilitated the functional studies described herein.

Secondary screening relied on enforced over expression of positiveclones in Jurkat T cells, and assaying for NF-AT activation. (Bram etal. (1993), supra) Jurkat T cells transiently transfected with thetagged-TACI-1 construct and an NFAT-reporter plasmid are incubated inmedium containing the monoclonal epitope-specific antibody. To maximizecross-linking of TACI-1, the antibodies are bound to beads beforeaddition to the cell suspensions. Following a 24 hour incubation, theactivity of the NFAT-reporter is determined. A dramatic induction ofNFAT reporter activity is found when cells are stimulated in thismanner. Control transfections without the TACI-1 construct do not showactivation following such treatment. Likewise, transfection of anunrelated cell surface molecule (CD8) followed by anti-CD8 stimulationdid not activate NFAT in these cells. The degree of activation was70-80% of the maximal stimulation that could achieved in these Jurkat Tcells by addition of phorbol ester plus ionomycin.

After screening a multiple tissue Northern blot (Clontech) with TACI-1cDNA (excised from the yeast two-hybrid vector), an independent TACI-1clone is obtained from a human fetal spleen cDNA library (Stratagene).The 5′-terminal coding region is confirmed by rapid amplification ofcDNA ends (RACE) using a ‘Marathon-ready’ human spleen cDNA library(Clontech), nested TACI-1-specific primers (5′-TCTGAATTGTTTTCAACTTCTC-3′(SEQ ID NO:9) and 5′-CAGCAGAGGATCCCAGTACTGCTC-3′ (SEQ ID NO:10)), andPfu polymerase (Stratagene) according to the manufacturers'recommendations.

Antisera.

cDNAs encoding the N-terminal 146 amino acid residues of CAML and theN-terminal 151 residues of TACI-1 are each cloned into a GST-fusionbacterial expression vector (Pharmacia). Rabbit polyclonal antisera areraised against purified GST-fusion proteins [Smith & Johnson (1988) Gene67:31-40], and specific antibodies are purified by immunoaffinitychromatography over the purified proteins coupled to agarose (Pierce),using standard techniques. Cross-linked anti-TACI-1 is prepared byincubating immunoaffinity-purified polygonal anti-TACI-1 antibodies withanti-rabbit IgG antibody-coupled magnetic beads (PerSeptiveDiagnostics).

Screen for Identifying Novel Immunosuppressant Drugs:

Jurkat T cells are transfected with TACI-1 expression plasmid and aNF-AT-reporter plasmid. Jurkat T cells naturally express the T-cellreceptor (TCR). The cells are stimulated by the addition of antibodiesto TACI-1, antibodies to TCR, or antibodies to both. Candidate drugs aremixed with the Jurkat T cells and the effect of these drugs isdetermined.

The NF-AT-reporter plasmid contains the SEAP reporter. This signal isused to measure the degree of inhibition of activation. The SEAPreporter assay is scaled up so as to be performed by a robot screeningapparatus. Drugs that block the activation of TACI-1 but not TCR asmeasured by the SEAP reporter assay are identified as having selectiveinhibition of the TACI-1 activated response.

Results and Discussion

Proteins that can interact with CAML are identified by using atwo-hybrid screen (Fields & Song (1989), supra); (Durfee et al. (1993),supra) with CAML as bait. To determine whether one of these identifiedCAML-binding proteins can affect Ca²⁺ signaling in T-cells, theirability to modulate the activity of the Ca²⁺-dependent transcriptionfactor NF-AT is examined [Truneh et al. (1985) Nature 313:318-321];[Verweij et al. (1990) J. Biol. Chem. 265:15788-15795]; [Karttunen &Shastri (1991) Proc. Natl. Acad. Sci. USA 88:3972-3976]; [Emmel et al.(1989) Science 246:1617-1620]. Enforced over expression of thetwo-hybrid clones in Jurkat T-cells reveals that one clone (encoding theTACI-1 protein), replaced the requirement for Ca²⁺ influx, implying thatTACI-1 lies in the same signal pathway as CAML. Northern blot analysisfor TACI-1 mRNA demonstrates a 1.4 kb mRNA expressed only in spleen,small intestine, thymus and peripheral blood lymphocytes suggesting alimited expression of TACI-1 (FIG. 1). The pattern observed isconsistent with the expression of TACI-1 being predominantly inperipheral blood cells, since peripheral blood cells, and in particularlymphocytes, can be present in all of these organs (including thePeyer's patches lining the small intestine.) Furthermore, there is nodetection of expression in colon, testis, ovary, or prostate. Inaddition, the TACI-1 protein is detected in all normal peripheral Blymphocytes using specific-antibody staining. There is no detectableprotein expressed in peripheral T-lymphocytes, monocytes or neutrophils.

Determination of the DNA sequence from both strands of the DNA isolatedreveals a complete open reading frame of 1325 base pairs, which ispredicted to encode a protein of 293 amino acids. The deduced amino acidsequence of TACI-1 (FIG. 2A) includes a single hydrophobic region(residues 167 to 186) (SEQ ID NO:8, which is encoded by the nucleic acidsequence provided in SEQ ID NO:7), that has features of a membranespanning segment (FIG. 2B). Analysis of the protein sequence by themethod of Sipos [Sipos & von Heijne (1993) Eur. J. Biochem.213:1333-1340]; [Claros & von Heijne (1994) Comput. Appl. Biosci.10:685-686], predicts extracellular exposure for the N-terminus andcytoplasmic exposure for the C-terminus. Although TACI-1 lacks anN-terminal signal sequence, the presence of an upstream stop codonindicates that the complete open reading frame is contained within theclone. TACI-1 is relatively rich in cysteine residues, but there is nosignificant sequence similarity or homology to any other disclosedprotein. A search for Prosite motifs in TACI-1 reveals one TNFR_NGFRmotif [Bairoch (1993) Nucleic Acids Res. 21:3097-3103] (residues 33-71)N-terminal to a putative transmembrane region, which consists ofC-x(4,6)-[FYH]-x(5,10)—C-x(0,2)—C-x(2,3)—C-x(7,11)—C-x(4,6)-[DNEQSKP]-x(2)-C(SEQ ID NO:11) in the N-terminal half of the protein. This motif isfound in a number of proteins, some of which are receptors for growthfactors. Some of these proteins have one copy of this motif. Acomparison of the TACI-1 protein sequence with itself reveals asignificant repeat between the TNFR_NGFR motif at residues 33-66 andresidues 70-104. This analysis drew attention to the presence of twoTNFR-type cysteine-rich domains encompassing these regions that indicatethat TACI-1 is a member of the superfamily of TNFR receptors. (FIG. 2C)

To confirm that TACI-1 is a transmembrane protein, its expression onJurkat T cells transfected with a TACI-1-encoding plasmid using flowcytometry was examined. Cells transfected with TACI-1 show surfacestaining with rabbit polyclonal antibodies raised against a fusionprotein that includes the N-terminal 12 kilodalton portion of TACI-1(FIG. 3A). Additional evidence that TACI-1 is localized at the cellsurface is derived from immunofluorescence microscopy, where surfacestaining of intact cells transfected with an N-terminalFLAG-epitope-tagged TACI-1 expression plasmid is observed (FIG. 3B).Since the N-terminus of TACI-1 is extracellular in the absence of acleaved signal sequence, it is a type III transmembrane protein[Wilson-Rawls et al., (1994) Virology 201:66-76].

To assess the effect of TACI-1 on NF-AT activity in T-cells, the proteinis transiently expressed in TAg Jurkat T cells with a secreted alkalinephosphatase reporter driven by the NF-AT-binding sequences from the IL-2promoter (Bram & Crabtree (1994), supra); [Fiering et al. (1990) GenesDev. 4:1823-1834]; (Bram et al. (1993), supra). TACI-1 over expressioncan partially replace the requirement for ionomycin in this assay. Theaddition of anti-TACI-1 antibodies to the cells further increased NF-ATactivation (more than twofold, see FIG. 4A), demonstrating that TACI-1responds to cross-linking at the cell surface. The degree of NF-ATactivation varies in different experiments due to transfectionefficiency but is typically 40-50% of the maximal response to thecorresponding treatment of the cells with PMA plus ionomycin.TACI-1-mediated NF-AT activation is dependent on calcineurin, as isdemonstrated by the loss of NF-AT activity in the presence of animmunosuppressive drugs, such as Cyclosporin A or FK506 [Friedman &Weissman (1991) Cell 66:799-806]; (Lui et al. (1991), supra) (FIG. 4B).

To examine the requirement for Ca²⁺ influx in TACI-1-mediated activationof NF-AT, extracellular calcium can be removed by the addition ofincreasing concentrations of EGTA. This results in the inhibition ofTACI-1-mediated NF-AT activation, as has been shown previously forT-cell receptor-mediated activation (FIG. 4C). As a control, the effectof expressing in the cells, a constitutively active, calcium-independentmutant of calcineurin A [Hubbard & Klee (1989) Biochemistry 28:1868-74];(O'Keefe et al. (1992), supra); (Clipstone & Crabtree (1993), supra) isexamined. As expected, in these cells NF-AT activation is seen even inthe presence of EGTA (FIG. 4C). Thus, in T-cells, TACI-1 mediates thecalcineurin-dependent aspect of the activation of NF-AT by initiatingthe influx of extracellular Ca²⁺ (most likely through the capacitativeCa²⁺ influx pathway following the depletion of intracellular stores[Putney & Bird (1993) Cell 75:199-201]; Hoth & Prenner (1993) Physiol.465:359-386]; [Zweifach & Lewis (1993) Proc. Natl. Acad. Sci. USA90:6295-6299]; [Premack et al. (1994) J. Immunol. 152:5226-5240).

Activation of NF-AT by CAML requires exogenous stimulation of proteinkinase C by the addition of phorbol ester (Bram & Crabtree (1994),supra). Antibody-cross linked TACI-1, however, is able to activate NF-ATin the absence of either PMA or ionomycin (FIG. 4B, solid bars).Experiments examining the activation of an AP-1 reporter by the overexpression of TACI-1 shows that AP-1 activation is elevated (overfour-fold) in TACI-1-transfected Jurkat T cells. This effect can befurther enhanced with the addition of cross-linked anti-TACI-1antibodies (FIG. 4D). Therefore, TACI-1 initiates Ca^(2t) influx, whichin turns activates calcineurin, as well as activates the AP-1 pathwayfollowing stimulation, thereby mediating the fulfillment of bothrequirements for the activation of NF-AT.

Further confirmation that TACI-1 interacts with CAML can be demonstratedby their specific interaction in a two-hybrid reverse swap experiment(Durfee et al. (1993), supra) (FIG. 5A). To define the critical aminoacid residues involved in the interaction, deletion mutants of bothTACI-1 and CAML are tested for their ability to physically associate(FIG. 5A). The C-terminal 126 amino acids of TACI-1 are found to besufficient for binding to the N-terminal 146 amino acids of CAML.Additional evidence for the in vivo association of TACI-1 with CAML isprovided by experiments in which full length CAML and a mutantcomprising the 146 N-terminal amino acid residues of CAML areco-immunoprecipitated with TACI-1 from cell lysates (FIG. 5B).Therefore, it may be concluded that the cytoplasmic C-terminal tail ofTACI-1 can physically associate with the N-terminal half of CAML.

To examine whether TACI-1 signaling depends on the association of TACI-1with CAML, the interacting domain of CAML (residues 1-146) is tested todetermine if it can inhibit TACI-1-induced NF-AT activation in adominant negative fashion. Co-transfection of the mutant CAML(1-146)expression plasmid completely eliminates TACI-1-induced NF-AT activationin Jurkat T cells. On the other hand, there is no inhibitory effect onPMA plus ionomycin-induced NF-AT activity, thus ruling out a nonspecifictoxic effect. Co-expression of CAML(1-146) also does not influence theaccumulation of TACI-1 protein as detected by Western blot analysis(FIG. 5C). CAML(1-146) lacks the hydrophobic transmembrane domains thatare required for Ca²⁺ influx activity [Holloway & Bram (1996) Biol.Chem. 271:8549-8552]. Hence the elimination of NF-AT-inducing activityin these cells can be attributed to binding of the CAML(1-146) fragmentto the intracellular C-terminal portion of TACI-1, preventingassociation with endogenous full-length CAML.

CAML is an integral membrane protein localized to cytoplasmic vesicles(Bram & Crabtree (1994), supra). Analysis of deletion mutants has shownthat hydrophobic domains in the C-terminal half of the protein areessential for activity (Holloway & Bram (1996), supra), and that thehydrophilic N-terminal half of the protein may have a regulatory role.Trypsin digestion experiments further demonstrated that the N-terminalhalf of the molecule is cytoplasmic. Here, the interaction betweenTACI-1 and CAML is required for TACI-1-mediated NF-AT activation inJurkat T cells is demonstrated. Taken together, these data indicate thata physical interaction between TACI-1 in the plasma membrane andintracellular CAML-containing vesicles can initiate a calcium influxsignal (FIG. 5D). These findings provide the first evidence for directcommunication between cell surface receptors and intracellularorganelles in lymphocytes. This mechanism may be somewhat analogous tothe dihydropyridine-ryanodine receptor model in muscle cells, in whichstimulation of one molecule can directly modulate the activity of theother [Marty et al. (1994) Proc. Natl. Acad. Sci. USA 91:2270-2274];[Sham et al. (1995) Proc. Natl. Acad. Sci. USA 92:121-125]; [Nakai etal. (1996) Nature 380:72-75].

Other cell surface proteins have been shown to activate lymphocytefunction, including the CD3 T-cell receptor, CD2, CD20, and Thy-1. Theseproteins have no sequence-homology with TACI-1, and it is likely thatthey play different roles from each other, either in terms of responseto different extracellular signals, and/or in terms of developmentalstage of expression on lymphocytes. TACI-1 must also play a role in themodulation of the function of lymphocytes in alternate and/orco-stimulatory pathways. Thus, in addition to defining a new signalingmechanism, TACI-1 is a novel lymphocyte-specific receptor capable ofactivating T-cells.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A method of treating an undesirable immune response in a mammal,comprising administering to the mammal a composition that comprises apolypeptide comprising amino acid residues 70-104 of SEQ ID NO: 2,wherein the undesirable immune response is selected from the groupconsisting of an autoimmune and inflammatory disease, a transplantationrejection, and a graft-versus host disease.
 2. The method of claim 1,wherein the polypeptide further comprises amino acid residues 33-66 ofSEQ ID NO:
 2. 3. The method of claim 1, wherein the polypeptidecomprises a chimeric protein.
 4. The method of claim 3, wherein thechimeric protein comprises an Fc domain of an immunoglobulin.
 5. Themethod of claim 4, wherein the chimeric protein comprises an Fc domainof an immunoglobulin.
 6. The method of claim 1, wherein the autoimmuneand inflammatory disease is selected from the group consisting of immunecomplex-induced vasculitis, glomerulonephritis, hemolytic anemia,myasthenia gravis, type II collagen-induced arthritis, experimentalallergic xenograft rejection, hyperacute xenograft rejection, rheumatoidarthritis, and systemic lupus erythematosus.
 7. The method of claim 6,wherein the autoimmune and inflammatory disease is myasthenia gravis. 8.The method of claim 6, wherein the autoimmune and inflammatory diseaseis type II collagen-induced arthritis.
 9. The method of claim 6, whereinthe autoimmune and inflammatory disease is rheumatoid arthritis.
 10. Themethod of claim 6, wherein the autoimmune and inflammatory disease issystemic lupus erythematosus.
 11. The method of claim 1, wherein themammal is human.
 12. A method of treating a lymphocyte cancer in amammal, comprising administering to the mammal a composition comprisinga polypeptide comprising amino acid residues 70-104 of SEQ ID NO: 2,wherein the lymphocyte cancer is selected from the group consisting ofmyeloma, lymphoma, and leukemia, and wherein administration of thesoluble TACI extracellular domain or the chimeric protein suppressescancer cell growth.
 13. The method of claim 12, wherein the polypeptidefurther comprises amino acid residues 33-66 of SEQ ID NO:
 2. 14. Themethod of claim 12, wherein the polypeptide comprises a chimericprotein.
 15. The method of claim 14, wherein the chimeric proteincomprises an Fc domain of an immunoglobulin.
 16. The method of claim 12,wherein the lymphocyte cancer is myeloma.
 17. The method of claim 12,wherein the lymphocyte cancer is lymphoma.
 18. The method of claim 12,wherein the lymphocyte cancer is leukemia.
 19. The method of claim 12,wherein the mammal is human.